JPH0431669B2 - - Google Patents
Info
- Publication number
- JPH0431669B2 JPH0431669B2 JP58020995A JP2099583A JPH0431669B2 JP H0431669 B2 JPH0431669 B2 JP H0431669B2 JP 58020995 A JP58020995 A JP 58020995A JP 2099583 A JP2099583 A JP 2099583A JP H0431669 B2 JPH0431669 B2 JP H0431669B2
- Authority
- JP
- Japan
- Prior art keywords
- particles
- immobilized
- biologically active
- hgg
- aminated
- 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 - Lifetime
Links
- 238000000034 method Methods 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 13
- 229940088623 biologically active substance Drugs 0.000 claims description 12
- 229920001577 copolymer Polymers 0.000 claims description 12
- 230000003100 immobilizing effect Effects 0.000 claims description 9
- 229920001282 polysaccharide Polymers 0.000 claims description 9
- 239000005017 polysaccharide Substances 0.000 claims description 9
- 125000003277 amino group Chemical group 0.000 claims description 7
- 239000011230 binding agent Substances 0.000 claims description 7
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 6
- 238000007254 oxidation reaction Methods 0.000 claims description 6
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 239000007857 degradation product Substances 0.000 claims description 3
- 229920000642 polymer Polymers 0.000 claims description 3
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims 1
- 239000002245 particle Substances 0.000 description 44
- 239000007790 solid phase Substances 0.000 description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 14
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 11
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 11
- 239000002953 phosphate buffered saline Substances 0.000 description 11
- 239000013543 active substance Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000000084 colloidal system Substances 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000006185 dispersion Substances 0.000 description 7
- 150000004804 polysaccharides Chemical class 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical class CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- -1 nickel peroxide Chemical class 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 229920000084 Gum arabic Polymers 0.000 description 3
- 241000978776 Senegalia senegal Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 235000010489 acacia gum Nutrition 0.000 description 3
- 239000000205 acacia gum Substances 0.000 description 3
- 230000004520 agglutination Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000007800 oxidant agent Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 235000010413 sodium alginate Nutrition 0.000 description 3
- 239000000661 sodium alginate Substances 0.000 description 3
- 229940005550 sodium alginate Drugs 0.000 description 3
- 159000000000 sodium salts Chemical class 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 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 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical class OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- ACKFDYCQCBEDNU-UHFFFAOYSA-J lead(2+);tetraacetate Chemical compound [Pb+2].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ACKFDYCQCBEDNU-UHFFFAOYSA-J 0.000 description 2
- 229920000620 organic polymer Polymers 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 150000003475 thallium Chemical class 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- MLIWQXBKMZNZNF-KUHOPJCQSA-N (2e)-2,6-bis[(4-azidophenyl)methylidene]-4-methylcyclohexan-1-one Chemical compound O=C1\C(=C\C=2C=CC(=CC=2)N=[N+]=[N-])CC(C)CC1=CC1=CC=C(N=[N+]=[N-])C=C1 MLIWQXBKMZNZNF-KUHOPJCQSA-N 0.000 description 1
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-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
- 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 1
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 102000015790 Asparaginase Human genes 0.000 description 1
- 108010074051 C-Reactive Protein Proteins 0.000 description 1
- 102100032752 C-reactive protein Human genes 0.000 description 1
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- JJLJMEJHUUYSSY-UHFFFAOYSA-L Copper hydroxide Chemical compound [OH-].[OH-].[Cu+2] JJLJMEJHUUYSSY-UHFFFAOYSA-L 0.000 description 1
- 239000005750 Copper hydroxide Substances 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000710799 Rubella virus Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102000011923 Thyrotropin Human genes 0.000 description 1
- 108010061174 Thyrotropin Proteins 0.000 description 1
- 241000589884 Treponema pallidum Species 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- ZYBXCIBWFOILPE-UHFFFAOYSA-N [Na].N#CC#N Chemical compound [Na].N#CC#N ZYBXCIBWFOILPE-UHFFFAOYSA-N 0.000 description 1
- XREOXKSDMNASCB-UHFFFAOYSA-N acetic acid;iodosylbenzene Chemical compound CC(O)=O.O=IC1=CC=CC=C1 XREOXKSDMNASCB-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium peroxydisulfate Substances [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 1
- VAZSKTXWXKYQJF-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)OOS([O-])=O VAZSKTXWXKYQJF-UHFFFAOYSA-N 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002391 anti-complement effect Effects 0.000 description 1
- 108010008730 anticomplement Proteins 0.000 description 1
- 229960003272 asparaginase Drugs 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- OZECDDHOAMNMQI-UHFFFAOYSA-H cerium(3+);trisulfate Chemical compound [Ce+3].[Ce+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O OZECDDHOAMNMQI-UHFFFAOYSA-H 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 description 1
- 229940011182 cobalt acetate Drugs 0.000 description 1
- QAHREYKOYSIQPH-UHFFFAOYSA-L cobalt(II) acetate Chemical compound [Co+2].CC([O-])=O.CC([O-])=O QAHREYKOYSIQPH-UHFFFAOYSA-L 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 108010018927 conglutinin Proteins 0.000 description 1
- 229910001956 copper hydroxide Inorganic materials 0.000 description 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 1
- 238000000354 decomposition reaction Methods 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
- 229920003045 dextran sodium sulfate Polymers 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- AFLBAXPZSPPPIW-UHFFFAOYSA-N disodium;dioxidoboranylformonitrile Chemical compound [Na+].[Na+].[O-]B([O-])C#N AFLBAXPZSPPPIW-UHFFFAOYSA-N 0.000 description 1
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
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- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
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- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 150000004678 hydrides Chemical class 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
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- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- QGRSLAYMCFPMHW-UHFFFAOYSA-M sodium;3-(2-methylprop-2-enoyloxy)propane-1-sulfonate Chemical compound [Na+].CC(=C)C(=O)OCCCS([O-])(=O)=O QGRSLAYMCFPMHW-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 125000003011 styrenyl group Chemical class [H]\C(*)=C(/[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
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The present invention relates to a method for immobilizing biologically active substances. Many methods are known for immobilizing biologically active substances such as enzymes, antigens, and antibodies onto solid supports. Among these, a simple and economical method is to immobilize glutaraldehyde on a carrier having an amino group using a binder. In order for this method to be applicable, it is necessary that the biologically active substance has an amino group, but since most biologically active substances such as enzymes, antigens, and antibodies have amino groups,
Immobilization on carriers is possible with glutaraldehyde. However, as a result of a detailed study of the method for immobilizing biologically active substances using the glutaraldehyde method, the present inventors found that biologically active substances immobilized by glutaraldehyde have properties that are undesirable for practical use. Something became clear. That is, proteins are more likely to be adsorbed to a solid phase in which a biologically active substance is immobilized using glutaraldehyde as a binder. Furthermore, when plasma or serum comes into contact with a solid phase on which a biologically active substance is immobilized using glutaraldehyde as a binding agent, complement is activated. These properties are unfavorable when a biologically active substance immobilized product, particularly an immunologically active substance immobilized product, is used for medical diagnosis or treatment. For example, in labeled immunoassays such as radioimmunoassays, enzyme immunoassays, and fluorescence immunoassays, the binding partner biological An objectively active substance is immobilized on a solid phase carrier and reacted with the immunoactive substance, but at this time, if the protein in the sample or the labeled immunoactive substance adsorbs nonspecifically to the solid phase, measurement accuracy will deteriorate. In addition, a method in which a particulate carrier immobilized with an immunologically active substance is reacted with a sample solution and the analyte is detected based on the presence or absence of agglutination is often used in immunoserological tests; Nonspecific aggregation caused by specific adsorption hinders testing. In addition, in recent years, a method of purifying blood by bringing immobilized substances such as immune-activating substances and enzymes into contact with blood or plasma outside the body to remove harmful components from the blood and then returning the blood or plasma to the body has been developed to treat intractable diseases. It is attracting attention as a new treatment method. However, in this case, if the complement in the blood is activated when the immobilized biologically active substance comes into contact with blood or plasma, there is a risk that the patient will suffer a shock. Considering the above circumstances, the present inventors investigated a method for immobilizing biologically active substances that overcomes the drawbacks while retaining the advantages of the glutaraldehyde method.
We have arrived at the present invention. The present invention uses a polysaccharide oxidation product as a binder and glycer (meth)acrylate alone or copolymerized as a carrier for immobilization on a biologically active carrier having an amino group. A method for immobilizing a biologically active substance, characterized by using an aminated product of a polymer, an aminated product of a single or copolymer of hydroxymethyl (meth)acrylate, or a Hofmann degradation product of a single or copolymer of acrylamide. It provides: The polysaccharide used in the method of the present invention may be neutral, acidic or basic, as long as the oxidation product produced by the oxidizing agent is water-soluble. Examples of suitable polysaccharides include dextran, dextran sodium sulfate, 1-amino-2-hydroxypropyldextran, starch, dextrin, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose and its sodium salt, gum arabic, alginic acid and Its sodium salt, heparin, chondroitin sulfate and its sodium salt, hyaluronic acid, etc. Examples of oxidizing agents for oxidizing polysaccharides include chromic acid, lead tetraacetate, periodic acid, hodosyl compounds (for example, iodosylbenzene acetate C 6 H 5 I (OCOCH 3 ) 2 ), and oxygen. (using cobalt acetate as a catalyst), peroxosulfuric acid-silver salt, copper chloride, copper hydroxide, thallium salt, thallium salt, acidic cerium sulfate, bismuthate, nickel peroxide, xenonic acid, anodic oxidation, and the like. In particular, periodic acid and lead tetraacetate are preferably used. As the periodic acid, free periodic acid may be used, or salts such as sodium periodate and potassium periodate may be used. The charging ratio of polysaccharide and oxidizing agent in the oxidation reaction of polysaccharide is preferably in the range of 1:2 to 200:1 by weight. In addition, the reaction temperature is 0 to 40â, and the pH is 2 to 40â.
10 is preferred. The carrier may be any solid having an amino group, and is not particularly limited, but organic polymers are particularly preferred from the viewpoint of ease of molding. The shape of the carrier is spherical, cylindrical,
Any material suitable for the purpose may be used, such as a disk, test tube, cylinder, fiber, membrane, particle, mesh, microtray, or spiral. Some examples of organic polymers preferable as carrier materials include aminated styrene alone or copolymers, aminated acrylic acid or methacrylic acid glycidyl esters alone or copolymers, and acrylonitrile alone. or a hydride of a copolymer, a Hofmann decomposition product of acrylamide alone or a copolymer, a hydrazide of an acrylic acid or a methacrylic acid alone or a copolymer, and the like. Among these, hydrophilic polymers are preferred, such as aminated products of glycer (meth)acrylate alone or copolymers, aminated products of hydroxyethyl (meth)acrylate alone or copolymers, and Hofmann degradation products of acrylamide alone or copolymers. products etc. are preferably used. When immobilizing a biologically active substance on a carrier, the carrier, the biologically active substance, and the polysaccharide periodate acetate may be mixed and reacted at the same time, but the biologically active substance In order to avoid a decrease in the activity of the carrier, it is preferable to first treat the carrier with periodate oxide of the polysaccharide and then contact it with the biologically active substance to immobilize it. Examples of biologically active substances that can be immobilized include enzymes such as asparaginase and urease, hormones such as human chorionic conadotropin, follicle-stimulating hormone, and thyroid-stimulating hormone, and antibodies against them, Treponema pallidum and A. Antigens of pathogens such as type or B hepatitis virus, rubella virus, toxplasma, mycoplasma, and streptococci and antibodies against them, cancer-related antigens such as α-fetoprotein and carcinoembryonic antigen and antibodies against them, ribonucleic acid and deoxyribonucleic acid and antibodies against it, immunoglobulins such as IgG, IgM, IgA, IgD, and IgE and antibodies against them, heat-agglutinated IgG,
Rheumatoid factor, C-reactive protein and its antibodies, CIqã»
CIrã»CIsã»C2ã»C3ã»C4ã»C5ã»C6ã»C7ã»C8ã»C9
These include complements and antibodies against them, various allergens, conglutins such as protein A, conglutinin, immunoconglutinin, and concanavalin, various histocompatibility antigens and antibodies against them. The present invention will be specifically explained below with reference to Examples. [Example 1] (Preparation of fluorescent colloid) Methyl methacrylate, glycidyl methacrylate, methacrylic acid, sulfopropyl methacrylate sodium salt, and ethylene glycol dimethacrylate were mixed in a molar ratio of 50:20:15:5:10, and 0.125%. These monomers were added to a sodium dodecyl sulfate (SDS) aqueous solution so that the total weight was 10%, and the monomers were emulsified by stirring vigorously under a nitrogen atmosphere. Polymerization was carried out at 60°C for about 1.5 hours using 1mM ammonium persulfate as an initiator. Next, unreacted monomers in the reaction solution were removed by extraction with carbon tetrachloride. The obtained colloidal particles with a diameter of approximately 0.15 Όm were heated at 30°C in a 0.025% sulfuric acid aqueous solution.
After hydrolysis for 1 hour, the mixture was neutralized with sodium hydroxide and washed. Washing was performed by centrifugation at 17500 rpm for 20 minutes. After washing, the colloidal particles were dispersed in dimethyl sulfoxide (DMSO) and
Aminated with 12% ammonia in DMSO. The reaction was carried out at 35°C for 3.5 hours, and ammonia was removed by thorough washing. Separately, gum arabic was dissolved in water to a concentration of 4 mg/ml and sodium periodate was dissolved in water to a concentration of 5 mg/ml (pH reached 3.0), and the mixture was stirred at 25°C for 1 hour. Aminated colloid particles were dispersed in this reaction solution to a concentration of 1%, the pH was adjusted to 10, and the mixture was stirred at 30° C. for 3 hours. After the reaction, excess gum arabic oxide was removed by centrifugal washing, and the colloidal particles were
Dispersed in DMSO to 0.1%, 0.2mg/ml
dichlorotriazinylaminofluorescein and
The reaction was carried out at 40°C for 1 hour. The obtained fluorescent colloid particles were washed with a 0.1M NaHCO 3 -NaOH buffer solution of pH 10.0, and then dispersed in the same buffer solution to a concentration of 0.1% and stored. (Immobilization of anti-HGG antibody to fluorescent colloid particles) 500 Ό of anti-HGG (human IgG) antibody IgG fraction (Miles) 240 Όg/ml solution prepared in goat and 500 Ό of fluorescent colloid particles for immobilizing 0.1% binding substance. The mixture was mixed and reacted at 4°C for 2 days. After the reaction, 15 mg of sodium cyanoborate was added and reacted at 25°C for 1 hour to reduce Schiff's base. The obtained anti-HGG antibody-bound colloid particles
It was washed with 0.1M Tris-HCl buffer at pH 8.0 and stored in the same buffer containing 1% bovine serum albumin (BSA). The anti-HGG antibody-immobilized colloid particles prepared as described above remained dispersed even after 4 weeks.
On the other hand, when anti-HGG antibodies were similarly immobilized on aminated colloids using glutaraldehyde,
The colloid agglomerated in about one week, making it difficult to use. (Preparation of particles for solid phase) Particles used as a solid phase were prepared by preparing glycidyl methacrylate, glycidyl methacrylate,
2-oxyethyl methacrylate and triethylene glycol dimethacrylate
It was prepared by aminating and hydrolyzing particles that were mixed and polymerized at a molar ratio of 87.5:9.5:4.8.
These are hydrophilic particles with an average particle size of 4.3 ÎŒm. (Immobilization of anti-HGG antibody to solid phase particles) Anti-HGG antiserum (Miles) was added to Protein A-
The IgG fraction was purified by Sepharose (Pharmacia) column chromatography. The IgG fraction of the prepared anti-HGG antiserum was dissolved in 0.15 molar phosphate buffered saline (PBS) at pH 7.2 containing 10 mg/ml of the anti-HGG antiserum to a concentration of 1%.
According to the method described in No. 141559, aminated solid phase particles activated with glutaraldehyde were dispersed and reacted at 25°C for 2 hours. Particles are washed by sedimentation using centrifugation at 3000 rpm, resulting in a concentration of 0.1%.
was dispersed in bovine serum albumin (abbreviated as BSA) to prepare anti-HGG antibody-immobilized solid-phase particles. (HGG measurement) HGG (Miles) 10 ÎŒm/ml to 1ng/ml
Prepare a series of double dilutions (90Ό each) and place a 1% anti-HGG antibody-immobilized solid phase microparticle dispersion in a glass test tube.
Incubate at 25°C for 1 hour while shaking with 100Ό
By centrifugation and redispersion at 3000 rpm, the solid phase particles were washed three times with Tris buffer, and HGG was
A solid phase bound via HGG antibody was obtained. 10Ό of a 0.02% dispersion of anti-HGG antibody-immobilized fluorescent colloidal particles and 100Ό of this 1% dispersion of solid phase particles were mixed in a test tube, reacted for 1 hour at 25°C, and the solid phase was thoroughly washed with Tris buffer. After washing, the solid phase particles were dispersed in 2 ml of Tris buffer, excited with 450 nm light, and the fluorescence intensity at 530 nm was measured. A Hitachi 650-40 fluorometer was used. The relationship between HGG concentration and fluorescence intensity in the range of 10 Όg/ml to 1 Όg/ml of HGG is shown in FIG. [Example 2] (Preparation of bovine serum albumin-immobilized particles) Aminated hydrophilic particles with an average diameter of 3.0 Όm prepared by the method described in JP-A-56-141559 were dispersed in distilled water at a concentration of 1%. . Separately, sodium periodate was added at a concentration of 1 mg/ml to an aqueous solution containing 1% sodium alginate, and the mixture was stirred at 25° C. for 1 hour to oxidize the sodium alginate. Next, 2 volumes of the aminated hydrophilic particle dispersion and 1 volume of the sodium alginate oxidation reaction solution were mixed, and the mixture was mixed with sodium hydroxide.
The pH was adjusted to 10 and stirred at 30°C for 30 minutes. The particles were then washed with PBS and dispersed in PBS at a concentration of 5%. 1 of the above particle dispersion (5%) and an equal volume of BSA
Mix the mg/ml PBS solution and stir at 30°C for 30 minutes.
BSA was immobilized on particles. BSA immobilized particles
After washing with PBS, the particles were dispersed in PBS supplemented with 1% human serum albumin so that the particle content was 1.25%. (Activity measurement of bovine serum albumin-immobilized particles) Anti-BSA antiserum (rabbit) (2.3 mg/ml) was diluted 10 times with PBS supplemented with 1% human serum albumin, and further diluted with 2n on a U-shaped microplate. A series of two-fold dilutions was made, and 25 Όl was added to each well. After standing still for 30 minutes, 25Ό of BSA-immobilized particle dispersion was added to each well, mixed, and left standing for 2 hours.
As a control experiment, the same procedure was performed using healthy rabbit serum instead of anti-BSA antiserum. Table 1 shows the results of observing the sedimentation image after standing for 2 hours.
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Dextran T70 and sodium periodate were both dissolved in distilled water to a concentration of 10 mg/ml and 2 mg/ml, respectively, and stirred at 25°C for 1 hour. The average particles used in Example 1 in this reaction solution
Aminated particles for solid phase of 4.3 Όm were dispersed at a concentration of 1%, the pH was adjusted to 9.6 with a small amount of sodium hydroxide and sodium hydrogen carbonate, and the mixture was stirred at 25° C. for 1 hour. After the reaction, the particles were washed with a pH 9.6 buffer solution (sodium hydroxide + sodium hydrogen carbonate), dispersed in a solution of IgG dissolved in the same buffer solution to a polymer concentration of 1%, and incubated at 25°C for 3 IgG was immobilized on the particles by stirring for a period of time. For comparison, IgG was similarly immobilized on solid-phase aminated particles using glutaraldehyde instead of periodic acid oxide in Textran. The IgG-immobilized particles were dispersed in a 1% sodium cyanogen borohydride solution (pH 4.0) (concentration 1%), stirred at 25°C for 30 minutes, and then washed with a Tris-HCl buffer solution (pH 7.5). It was dispersed in PBS at a concentration of 1% and stored in a refrigerator.
The amount of IgG immobilized on the particles was determined by hydrolysis with hydrochloric acid and amino acid analysis. The presence or absence of complement activation by the IgG-immobilized particles was tested as follows. That is, a 1% particle dispersion and an equal volume of fresh human serum were mixed and incubated at 37â for 15 minutes.
I left it for a minute. Next, after precipitating the particles with PBS, add 1
Disperse again on a glass slide at a concentration of %
The mixture was mixed with anti-complement and anti-serum (rabbit) diluted 100 times with PBS, and the presence or absence of agglutination was determined visually.
The results are summarized in Table 2.
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å°ã§ããã[Table] However, healthy rabbit serum was always used as a control when determining agglutination. As is clear from Table 2, when IgG was immobilized using glutaraldehyde as a binding agent, activated complement components were bound to the solid phase, but when dextran oxide was used as a binding agent, the activated complement components were bound to the solid phase. There is no or minimal binding of complement components to the solid phase.
第ïŒå³ã¯å®æœäŸïŒã®HGG枬å®çµæã瀺ãå³ã§
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FIG. 1 is a diagram showing the HGG measurement results of Example 1.
Claims (1)
ããåºãæããæ äœã«åºå®åããã«ããã€ãŠãçµ
åå€ãšããŠå€ç³é¡ã®é žåçæç©ããæ äœãšããŠã°
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äœã®ã¢ããåç©ãããããã·ã¡ãã«ïŒã¡ã¿ïŒã¢ã¯
ãªã¬ãŒãã®åç¬ãŸãã¯å ±éåäœã®ã¢ããåç©ããŸ
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ãã³å解çæç©ãçšããããšãç¹åŸŽãšããçç©åŠ
ç掻æ§ç©è³ªã®åºå®åæ³ã1. When immobilizing a biologically active substance having an amino group on a carrier having an amino group, an oxidation product of a polysaccharide is used as a binder and glycer (meth)acrylate alone or copolymerized as a carrier. A method for immobilizing a biologically active substance, characterized by using an aminated product of a polymer, an aminated product of a single or copolymer of hydroxymethyl (meth)acrylate, or a Hofmann degradation product of a single or copolymer of acrylamide. .
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2099583A JPS59146589A (en) | 1983-02-10 | 1983-02-10 | Immobilization of biologically active substance |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2099583A JPS59146589A (en) | 1983-02-10 | 1983-02-10 | Immobilization of biologically active substance |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS59146589A JPS59146589A (en) | 1984-08-22 |
JPH0431669B2 true JPH0431669B2 (en) | 1992-05-27 |
Family
ID=12042698
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2099583A Granted JPS59146589A (en) | 1983-02-10 | 1983-02-10 | Immobilization of biologically active substance |
Country Status (1)
Country | Link |
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JP (1) | JPS59146589A (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JPH0387662A (en) * | 1989-08-31 | 1991-04-12 | Tokai Univ | Plastic plate for biochemical measurement and production thereof and biochemical measure method using this plate |
CN102732500B (en) * | 2012-07-04 | 2013-09-04 | æµæ±åæå€§åŠ | Preparation method of oxidized dialdehyde cellulose immobilized urease |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5456895A (en) * | 1977-09-19 | 1979-05-08 | Hoffmann La Roche | Novel latex polymer and method of producing same |
-
1983
- 1983-02-10 JP JP2099583A patent/JPS59146589A/en active Granted
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5456895A (en) * | 1977-09-19 | 1979-05-08 | Hoffmann La Roche | Novel latex polymer and method of producing same |
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