EP0000772A1 - Immunological reagent consisting of special latex particles based on a vinylpolymer covered with an immunologically active material; process for its preparation; use of this reagent; analysing process using this reagent and test-kit containing this reagent - Google Patents
Immunological reagent consisting of special latex particles based on a vinylpolymer covered with an immunologically active material; process for its preparation; use of this reagent; analysing process using this reagent and test-kit containing this reagent Download PDFInfo
- Publication number
- EP0000772A1 EP0000772A1 EP78100581A EP78100581A EP0000772A1 EP 0000772 A1 EP0000772 A1 EP 0000772A1 EP 78100581 A EP78100581 A EP 78100581A EP 78100581 A EP78100581 A EP 78100581A EP 0000772 A1 EP0000772 A1 EP 0000772A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- reagent
- latex
- particles
- immunologically active
- sample
- 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.)
- Withdrawn
Links
- 229920000126 latex Polymers 0.000 title claims abstract description 79
- 239000004816 latex Substances 0.000 title claims abstract description 76
- 239000002245 particle Substances 0.000 title claims abstract description 50
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 41
- 239000011149 active material Substances 0.000 title claims abstract description 26
- 229920002554 vinyl polymer Polymers 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 19
- 230000001900 immune effect Effects 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 229920000642 polymer Polymers 0.000 claims abstract description 39
- 239000013543 active substance Substances 0.000 claims abstract description 15
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims abstract description 13
- -1 lipoids Chemical class 0.000 claims abstract description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 8
- 150000001993 dienes Chemical class 0.000 claims abstract description 5
- 239000006185 dispersion Substances 0.000 claims abstract description 3
- 239000000178 monomer Substances 0.000 claims description 43
- 230000004520 agglutination Effects 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 15
- 229920001577 copolymer Polymers 0.000 claims description 14
- 229940098197 human immunoglobulin g Drugs 0.000 claims description 11
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 9
- 230000008105 immune reaction Effects 0.000 claims description 9
- 229940027941 immunoglobulin g Drugs 0.000 claims description 8
- 229920001519 homopolymer Polymers 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 6
- 108010088751 Albumins Proteins 0.000 claims description 5
- 102000009027 Albumins Human genes 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 5
- 239000007900 aqueous suspension Substances 0.000 claims description 5
- 241000283707 Capra Species 0.000 claims description 4
- 230000001588 bifunctional effect Effects 0.000 claims description 4
- 238000006193 diazotization reaction Methods 0.000 claims description 4
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 claims description 3
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 3
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 3
- GYCMBHHDWRMZGG-UHFFFAOYSA-N Methylacrylonitrile Chemical compound CC(=C)C#N GYCMBHHDWRMZGG-UHFFFAOYSA-N 0.000 claims description 3
- 125000005250 alkyl acrylate group Chemical group 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- OEPOKWHJYJXUGD-UHFFFAOYSA-N 2-(3-phenylmethoxyphenyl)-1,3-thiazole-4-carbaldehyde Chemical compound O=CC1=CSC(C=2C=C(OCC=3C=CC=CC=3)C=CC=2)=N1 OEPOKWHJYJXUGD-UHFFFAOYSA-N 0.000 claims description 2
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 claims description 2
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 claims description 2
- XUGUHTGSMPZQIW-UHFFFAOYSA-N [[4-(4-diazonioiminocyclohexa-2,5-dien-1-ylidene)cyclohexa-2,5-dien-1-ylidene]hydrazinylidene]azanide Chemical compound C1=CC(N=[N+]=[N-])=CC=C1C1=CC=C(N=[N+]=[N-])C=C1 XUGUHTGSMPZQIW-UHFFFAOYSA-N 0.000 claims description 2
- JXLHNMVSKXFWAO-UHFFFAOYSA-N azane;7-fluoro-2,1,3-benzoxadiazole-4-sulfonic acid Chemical compound N.OS(=O)(=O)C1=CC=C(F)C2=NON=C12 JXLHNMVSKXFWAO-UHFFFAOYSA-N 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 10
- 102000036639 antigens Human genes 0.000 abstract description 10
- 108091007433 antigens Proteins 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 abstract description 7
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 102000004895 Lipoproteins Human genes 0.000 abstract description 2
- 108090001030 Lipoproteins Proteins 0.000 abstract description 2
- 229930182558 Sterol Natural products 0.000 abstract description 2
- 229930013930 alkaloid Natural products 0.000 abstract description 2
- 150000001412 amines Chemical class 0.000 abstract description 2
- 150000001413 amino acids Chemical class 0.000 abstract description 2
- 238000004132 cross linking Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 abstract description 2
- 150000004676 glycans Chemical class 0.000 abstract description 2
- 239000005556 hormone Substances 0.000 abstract description 2
- 229940088597 hormone Drugs 0.000 abstract description 2
- 102000039446 nucleic acids Human genes 0.000 abstract description 2
- 108020004707 nucleic acids Proteins 0.000 abstract description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 2
- 239000005017 polysaccharide Substances 0.000 abstract description 2
- 229920001282 polysaccharide Polymers 0.000 abstract description 2
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 2
- 150000003431 steroids Chemical class 0.000 abstract description 2
- 150000003432 sterols Chemical class 0.000 abstract description 2
- 235000003702 sterols Nutrition 0.000 abstract description 2
- 239000011782 vitamin Substances 0.000 abstract description 2
- 229940088594 vitamin Drugs 0.000 abstract description 2
- 229930003231 vitamin Natural products 0.000 abstract description 2
- 235000013343 vitamin Nutrition 0.000 abstract description 2
- 238000006116 polymerization reaction Methods 0.000 description 26
- 239000000872 buffer Substances 0.000 description 14
- 239000003995 emulsifying agent Substances 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 239000003999 initiator Substances 0.000 description 10
- 239000002253 acid Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 150000007513 acids Chemical class 0.000 description 6
- 235000014113 dietary fatty acids Nutrition 0.000 description 6
- 239000000194 fatty acid Substances 0.000 description 6
- 229930195729 fatty acid Natural products 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 150000004665 fatty acids Chemical class 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000012429 reaction media Substances 0.000 description 5
- 238000010998 test method Methods 0.000 description 5
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 238000000402 conductometric titration Methods 0.000 description 4
- 238000002405 diagnostic procedure Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 125000000129 anionic group Chemical group 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000012986 chain transfer agent Substances 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000003792 electrolyte Substances 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000007720 emulsion polymerization reaction Methods 0.000 description 3
- 239000007986 glycine-NaOH buffer Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 3
- 229920000151 polyglycol Polymers 0.000 description 3
- 239000010695 polyglycol Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 3
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- JAHNSTQSQJOJLO-UHFFFAOYSA-N 2-(3-fluorophenyl)-1h-imidazole Chemical compound FC1=CC=CC(C=2NC=CN=2)=C1 JAHNSTQSQJOJLO-UHFFFAOYSA-N 0.000 description 2
- VRVRGVPWCUEOGV-UHFFFAOYSA-N 2-aminothiophenol Chemical compound NC1=CC=CC=C1S VRVRGVPWCUEOGV-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical compound CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 2
- CNCOEDDPFOAUMB-UHFFFAOYSA-N N-Methylolacrylamide Chemical compound OCNC(=O)C=C CNCOEDDPFOAUMB-UHFFFAOYSA-N 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 239000003513 alkali Substances 0.000 description 2
- 239000000538 analytical sample Substances 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 2
- MPMBRWOOISTHJV-UHFFFAOYSA-N but-1-enylbenzene Chemical class CCC=CC1=CC=CC=C1 MPMBRWOOISTHJV-UHFFFAOYSA-N 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
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- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 2
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
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- LVHBHZANLOWSRM-UHFFFAOYSA-N methylenebutanedioic acid Natural products OC(=O)CC(=C)C(O)=O LVHBHZANLOWSRM-UHFFFAOYSA-N 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
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- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- QROGIFZRVHSFLM-UHFFFAOYSA-N prop-1-enylbenzene Chemical class CC=CC1=CC=CC=C1 QROGIFZRVHSFLM-UHFFFAOYSA-N 0.000 description 2
- HJWLCRVIBGQPNF-UHFFFAOYSA-N prop-2-enylbenzene Chemical compound C=CCC1=CC=CC=C1 HJWLCRVIBGQPNF-UHFFFAOYSA-N 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
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- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
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- SZHIIIPPJJXYRY-UHFFFAOYSA-M sodium;2-methylprop-2-ene-1-sulfonate Chemical compound [Na+].CC(=C)CS([O-])(=O)=O SZHIIIPPJJXYRY-UHFFFAOYSA-M 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical class [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000001124 (E)-prop-1-ene-1,2,3-tricarboxylic acid Substances 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- XHZPRMZZQOIPDS-UHFFFAOYSA-N 2-Methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid Chemical compound OS(=O)(=O)CC(C)(C)NC(=O)C=C XHZPRMZZQOIPDS-UHFFFAOYSA-N 0.000 description 1
- YYYOQURZQWIILK-UHFFFAOYSA-N 2-[(2-aminophenyl)disulfanyl]aniline Chemical compound NC1=CC=CC=C1SSC1=CC=CC=C1N YYYOQURZQWIILK-UHFFFAOYSA-N 0.000 description 1
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- KFFUEVDMVNIOHA-UHFFFAOYSA-N 3-aminobenzenethiol Chemical compound NC1=CC=CC(S)=C1 KFFUEVDMVNIOHA-UHFFFAOYSA-N 0.000 description 1
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- 238000007334 copolymerization reaction Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- LDHQCZJRKDOVOX-NSCUHMNNSA-N crotonic acid Chemical compound C\C=C\C(O)=O LDHQCZJRKDOVOX-NSCUHMNNSA-N 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- RZMWTGFSAMRLQH-UHFFFAOYSA-L disodium;2,2-dihexyl-3-sulfobutanedioate Chemical compound [Na+].[Na+].CCCCCCC(C([O-])=O)(C(C([O-])=O)S(O)(=O)=O)CCCCCC RZMWTGFSAMRLQH-UHFFFAOYSA-L 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002432 hydroperoxides Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000002734 metacrylic acid derivatives Chemical class 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 150000001451 organic peroxides Chemical class 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000004304 potassium nitrite Substances 0.000 description 1
- 235000010289 potassium nitrite Nutrition 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- DEWNCLAWVNEDHG-UHFFFAOYSA-M sodium;2-(2-methylprop-2-enoyloxy)ethanesulfonate Chemical compound [Na+].CC(=C)C(=O)OCCS([O-])(=O)=O DEWNCLAWVNEDHG-UHFFFAOYSA-M 0.000 description 1
- BWYYYTVSBPRQCN-UHFFFAOYSA-M sodium;ethenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C=C BWYYYTVSBPRQCN-UHFFFAOYSA-M 0.000 description 1
- YIVJSMIYMAOVSJ-UHFFFAOYSA-N sodium;phosphono dihydrogen phosphate Chemical compound [Na+].OP(O)(=O)OP(O)(O)=O YIVJSMIYMAOVSJ-UHFFFAOYSA-N 0.000 description 1
- 239000002195 soluble material Substances 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 description 1
- GTZCVFVGUGFEME-UHFFFAOYSA-N trans-aconitic acid Natural products OC(=O)CC(C(O)=O)=CC(O)=O GTZCVFVGUGFEME-UHFFFAOYSA-N 0.000 description 1
- LDHQCZJRKDOVOX-UHFFFAOYSA-N trans-crotonic acid Natural products CC=CC(O)=O LDHQCZJRKDOVOX-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000000007 visual effect Effects 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/823—Immunogenic carrier or carrier per se
Definitions
- the present invention relates to valuable diagnostic reagents, a process for their preparation and diagnostic methods in which such reagents are used.
- An antigen is a foreign substance which, when applied to the living being, causes the formation of certain soluble substances, known as antibodies. Any substance such as a protein which is not normally present in a particular living being can cause the formation of antibodies if it is applied to the living being under suitable conditions.
- the antibodies After their formation, the antibodies react with the antigens and in this way protect against infections in the case of a bacterial or virus foreign body.
- Immunological test methods are based on the antigen-antibody reaction, which usually manifests itself through insolubility or agglutination.
- an antigen or an antibody is confirmed or determined by adding the corresponding antibody or antigen to a body fluid of the living being, usually urine, blood serum or a specially treated blood extract.
- a body fluid of the living being usually urine, blood serum or a specially treated blood extract.
- other body fluids can also be used.
- the presence or absence of the antibody or the antigen in the body fluid of the living being is determined by determining the occurrence or non-occurrence of an antigen-antibody reaction.
- the antibody or antigen was' by means of a carbodiimide via an amide bond to discrete particles of carboxylated latex polymers, such as e.g. of carboxylated copolymers of butadiene and styrene.
- the present invention now relates to novel polymeric carriers with which the above disadvantages can be avoided and which with a wide range of immunologically active Materials can form a diagnostically usable reagent that is stable, specific, and sensitive, and that enables an easily detectable visual assessment in a very short time.
- the present invention relates to a water-insoluble reagent for immunological determination with a specific weight approximately equal to that of water in the form of discrete latex particles to which an immunologically active material is bound, characterized in that the latex consists of a dispersion of particles of vinyl polymers which as end groups are groups of the formula: wear, the particles of a core of vinyl and / or diene polymer which carries carboxyl and / or sulfonate functions, and of an outer layer of vinyl polymer which, as end groups, groups of the formula: carries, are formed and have an average diameter between 0.03 and 5 pm.
- the invention further relates to a method for producing such a reagent, which is characterized in that the latex is reacted with the immunologically active material after diazotization or in the presence of suitable bifunctional reagents.
- Immunologically active substances include amines, amino acids, peptides, proteins, lipoproteins, glycoproteides, sterols, steroids, lipoids, nucleic acids, enzymes, hormones, vitamins, polysaccharides and alkaloids.
- Preferred Immunologically active substances are listed in the following table:
- immunologically active substances are albumin, rheumatoid factor, human immunoglobulin IgG and antibodies against IgG.
- Vinyl polymers which form the core of the particles are understood to mean homopolymers of monomers, such as styrene and its derivatives: methylstyrenes, ethylstyrenes, vinyltoluene; Vinyl chloride, vinylidene chloride; Vinyl acetate; Acrylic derivatives, such as alkyl acrylates and methacrylates (alkyl having 1 to 10 carbon atoms) which are optionally hydroxylated, such as 2-hydroxyethyl acrylate and methacrylate and 2-hydroxypropyl acrylate and methacrylate; Acrylonitrile and methacrylonitrile; and copolymers of these monomers with one another and / or with modifying vinyl comonomers such as divinylbenzene, acrylamide and methacrylamide and their N-substituted derivatives, such as e.g. Methylolacrylamide; these comonomers represent up to 5% by weight of the copolymer.
- monomers
- Diene polymers that form the core are homopolymers of butadiene and its derivatives: chloroprene, isoprene; and the copolymers of these monomers with one another and / or with vinyl monomers, as mentioned above, in all proportions and / or with modifying vinyl monomers, as enumerated above, the amount of which in the copolymer makes up to 5% by weight.
- the vinyl polymers which form the outer layer of the particles are homopolymers of monomers, such as styrene and its derivatives, for example methylstyrenes, ethylstyrenes and vinyl toluene; optionally hydroxylated alkyl acrylates and alkyl methacrylates (alkyl with 1 to 10 carbon atoms); Acrylonitrile and methacrylonitrile; and copolymers of these monomers with one another and / or with modifying vinyl comonomers, such as divinylbenzene, acrylamide and methacrylamide, and their N-substituted derivatives, such as methylolacrylamide, which can make up up to 5% by weight of the copolymer.
- monomers such as styrene and its derivatives, for example methylstyrenes, ethylstyrenes and vinyl toluene
- the core polymer in the particles constitutes 30 to 99.5% by weight, preferably 60 to 99% by weight, and the polymer in the outer layer 70 to 0.5% by weight, preferably 40 to 1% by weight, represents.
- the polymer particles the particle size distribution of which may be wide or narrow depending on the desired properties of the latex and the applications under consideration, have an average diameter between 0.03 and 5 ⁇ m, preferably between 0.05 and 1 ⁇ m. They represent up to 60% by weight, preferably up to 45% by weight, of the latex. However, the latex can easily be diluted or concentrated.
- the core polymer can be prepared by emulsion polymerization of the vinyl monomer (s) and / or diene monomer (s) in the presence of at least one ethylenic mono- or polycarboxylic acid which is copolymerizable with the monomer (s) and / or at least one copolymerizable unsaturated alkali metal organosulfonate; then the polymer of the outer layer is prepared by emulsion polymerizing the vinyl monomer (s) in the presence of the latex of the core polymer obtained above and in the presence of a chain transfer agent.
- the monomers used in the polymerization of the core polymer and in the polymerization of the polymer of the outer layer are the monomers listed above. They are either all used before the polymerization or for Part used before the polymerization, the remaining part being added to the reaction medium in successive fractions or continuously in the course of the polymerization, or all added in successive fractions or continuously in the course of the polymerization.
- copolymerizable ethylenic mono- or polycarboxylic acids acrylic acid, methacrylic acid, maleic acid, fumaric acid, crotonic acid, sorbic acid, cinnamic acid, itaconic acid, aconitic acid may be mentioned, in amounts between 0.5 and 15% by weight, preferably between 0.5 and 10% by weight. -%, based on the monomer or monomers used.
- the copolymerizable unsaturated alkali organosulfonates are e.g. Sodium vinyl sulfonate, sodium methallyl sulfonate, sodium 2-sulfoethyl acrylate, sodium 2-sulfoethyl methacrylate, 2-acrylamido-2-methylpropane sulfonate; they are used in amounts between 0.1 and 3% by weight, based on the monomer or monomers.
- copolymerizable ethylene mono- or polycarboxylic acids and the copolymerizable unsaturated alkali organosulfonates can be used individually or in combination in the amounts specified.
- the core polymer is prepared in emulsion by any conventional method in the presence of an initiator and an emulsifier.
- the initiator used is preferably alkali metal sulfates, water-soluble diazo derivatives or redox systems based on hydrogen peroxide, organic peroxides or hydroperoxides in amounts of the order of 0.01 to 5% by weight, preferably 0.03 to 3% by weight, based on the or the monomers.
- the emulsifier used can be anionic and / or nonionic. These are classic products for emulsion polymerization.
- Salts of fatty acids may be mentioned as anionic emulsifiers; Alkaline alkyl sulfates, Alkalialkylsulfonate, Alkalialkylarylsulfonate, Alkalialkylsulfosuccinate, Alkalialkylphosphate; Alkyl sulfosuccinate; Sulfonates of alkylphenol polyglycol ethers; Salts of esters of alkylsulfopolycarboxylic acids; Condensation products of fatty acids with oxyalkanesulfonic acids and aminoalkanesulfonic acids; sulfated derivatives of polyglycol ethers; sulfated esters of fatty acids and polyglycols; Alkanolamides of sulfated fatty acids.
- Suitable nonionic emulsifiers are fatty acid esters of polyalcohols, alkanolamides of fatty acids, polyethylene oxides, copolyethylene oxide / propylene oxide and oxyethylated alkylphenols.
- the amounts of the emulsifier or emulsifiers to be used are of the order of magnitude of 0.01 to 5% by weight, based on the monomer or monomers, and they are introduced either in total before the polymerization or in part before the polymerization, the remaining part is added in the course of the polymerization in successive fractions or continuously to the reaction medium, or overall in the course of the polymerization in successive fractions or continuously.
- the amount of water to be used for the polymerization of the core polymer must be such that the concentration of the monomer or monomers does not exceed 60% by weight.
- any compounds to the reaction medium which are capable of modifying either the ionic strength of the medium and consequently the particle size distribution, such as mineral salts, electrolytes, in an amount of up to 3% by weight, based on the Monomers, or which can modify the pH of the medium, such as buffers, acids, bases.
- the medium is neutral or acidic.
- the polymerization temperature which is a function of the initiator used and the polymer to be prepared, is generally between -5 and + 90 ° C.
- the latices obtained have polymer particles with a diameter between 0.03 and 5 ⁇ m, preferably between 0.05 and 1 ktm. These particles are generally not calibrated, but it is possible to obtain them calibrated using known calibration methods for emulsion polymerization, such as the controlled addition of the emulsifier and / or the monomer (s) and in particular the inoculation. In the latter case, the emulsifier can be contained in the inoculum.
- the particles are formed from homopolymer or copolymer with a surface of carboxyl and / or sulfonate functions. The presence of these functions can be confirmed by conductometric titration.
- the preparation of the polymer of the outer layer is carried out in an aqueous emulsion in the presence of the core polymer, chain transfer agent, initiator and, if appropriate, emulsifier.
- the amount of the core polymer used is between 30 and 99.5% by weight and is preferably 60 to 99% by weight, based on the sum of the core polymer and the monomer or monomers to be polymerized.
- the chain transfer agent of the aminophenyl disulfide or aminophenyl mercaptan type is in particular o, o'-dithiobisaniline, p, p'-dithiobisaniline, 2-mercaptoaniline, 3-mercaptoaniline, 4-mercaptoaniline.
- This remedy is generally in solution used in the monomer or monomers, in amounts between 0.1 and 10% by weight, preferably between 0.5 and 5% by weight, based on the monomer or monomers.
- the initiators required for the polymerization of the outer layer or monomers are diazo initiators, azonitriles such as azo-bis-isobutyronitrile or such as sulfonated azonitriles as described in French Patent No.
- azobis isobutyronitrile sodium sulfonate
- azobis a-methylbutyronitrile sodium sulfonate
- azobis a-methyl- ⁇ -ethoxycarbonylbutyronitrile sodium sulfonate
- carboxylated azonitriles such as 4,4'-azobis (4-cyanopentanoic acid) and their salts
- azobis-alkylamidinium salts such as a, a'-azobis-isobutyramidinium chloride, azobis-N, N'-dimethylene-isobutyramidinium chloride.
- the initiator which is used in an amount of 0.01 to 3% by weight, preferably 0.1 to 2% by weight, based on the monomer or monomers, is used in whole or in part before the polymerization, where the other part is added to the reaction medium in successive fractions or continuously in the course of the polymerization, in particular if the life of the initiator at the polymerization temperature is short.
- the initiator can also be added continuously to the reaction medium overall in the course of the polymerization.
- the emulsifier if any, is selected from anionic and / or nonionic emulsifiers which have been specified for the preparation of the core polymer; it can be the same or different from the emulsifier used for the production of the core polymer. It is used in amounts of up to 10% by weight, based on the monomer or monomers, and depending on the average diameter of the latex particles to be obtained, it can be introduced either entirely before the polymerization or partly before the polymerization take place, the remaining part in the course of the polymer tion is added in successive fractions or continuously, or it can be done in the course of the polymerization in successive fractions or continuously.
- the amount of water to be used in the polymerization of the outer layer must be such that the concentration of core polymer and monomers to be polymerized or polymerized does not exceed 60% by weight, preferably 45% by weight.
- the polymerization temperature which is a function of the chosen initiator, is generally between 5 and 100 ° C, preferably between 40 and 90 ° C.
- the latices obtained have polymerization particles whose diameter is between 0.03 and 5 ⁇ m, preferably between 0.05 and 1 ⁇ m; since the amount of the outer layer is not very large, it does not noticeably modify the size of the particles of the core polymer.
- the particles may or may not be calibrated, but in certain applications it is preferred for reasons of reproducibility that they are calibrated, i.e. that they have a narrow grain size distribution.
- the latices are mechanical and resistant to storage and electrolytes, i.e. they do not flocculate if you are given mineral salts such as the chlorides, nitrates, borates, phosphates of sodium, calcium, magnesium, potassium are added.
- the particles are formed from polymers and have a surface with carboxyl and / or sulfonate functions and groups of the formula: on.
- the outer layer is polymerized on the core polymer, the carboxyl and / or sulfonate functions remain accessible, as can be shown by conductometric titration, and the groups of the formula: are available for further reactions.
- the immunologically active materials can be bound physically and / or chemically to the latex polymers used according to the invention.
- the reagent according to the invention is produced by forming an azo bond between the latex and the immunologically active material.
- the primary aromatic amino groups of the latex are converted into a diazonium salt.
- an inorganic acid such as hydrochloric acid, sulfuric acid or perchloric acid can be used.
- Sodium nitrite or potassium nitrite is preferably used as the nitrite.
- the reaction is preferably carried out at 0-5 C because of the instability of the diazonium salts.
- the immunologically active material is then reacted with the diazotized carrier in an aqueous medium, preferably between 0.degree. C. and room temperature.
- the immunologically active material can be bound to the latex used according to the present invention by means of a polyfunctional compound via an intermediate piece.
- polyfunctional compounds are useful ones which react with the aromatic amino groups of the latex polymer or undergo a substitution reaction at the aromatic ring of the latex polymer and at the same mino- with functional groups of the immunologically active material such as A, mercapto, carbonyl and hydroxyl groups react or undergo a substitution reaction on the aromatic ring of the immunologically active material.
- Representative representatives of such polyfunctional compounds are azó, isocyano, isothiocyano or aldehyde group-containing compounds such as e.g. Bis-diazobenzidine, bis-diazobenzidine-disulfonic acid, bis-diazo-p-phenyl diamine, phenyl diisocyanate, toluene diisocyanate, glutardialdehyde.
- the immunologically active material When reacting in the presence of a bifunctional compound, the immunologically active material is reacted with the carrier in an aqueous medium, preferably at room temperature (20 ° C. to 25 ° C.). However, the temperature can also be between 0 ° C and 40 ° C.
- the amount of the bifunctional compound used depends on the number of amino groups on the latex. A ten- to hundred-fold molar excess of the bifunctional compound compared to the number of amino groups of the latex used is preferably used.
- the pH of the reaction is important. It should not be chosen to denature a protein reactant. Usually the pH is between 5 and 9. This pH is determined using suitable ones. Buffer systems such as phosphate buffers and the like are maintained.
- the final product is a water-soluble material which is suspended in an aqueous buffer solution of pH 5.0 to 9, the pH of the solution being different from that in detail used system and depends on the requirements for the stability of the immunologically active material.
- the specific weight of the product corresponds approximately to that of water (0.97-1.02), whereby a stable suspension of the product is achieved.
- the products can be isolated, for example, by centrifugation in the form of a white or yellowish precipitate.
- the amount of immunologically active material which is bound to the immunologically inert latex polymer carrier is usually 0.01 to 15.0% by weight. However, each individual immunologically active material is used in an amount which is most useful in a diagnostic test. For this reason, each material is combined with the carrier in a ratio that best suits the specific requirements.
- the present invention therefore encompasses the use of such an amount of immunologically active material in combination with an immunologically inert latex polymer carrier which is suitable for providing a reagent useful for such diagnostic purposes.
- the product can be used in specific diagnostic tests based on immunological principles.
- the determination of the immunologically active substance can be carried out both in a direct and in an indirect (inhibition) test method.
- the analytical sample and the latex particles coated with the corresponding immunological reaction partner are mixed to determine an immunologically active substance and the occurrence of agglutination is observed.
- the test is positive if agglutination is detected.
- the analytical sample is mixed with a certain amount of the corresponding immunological reaction partner (e.g. antiserum) and latex particles coated with the immunologically active substance to determine an immunologically active substance and the occurrence of agglutination is observed.
- the test is positive if no agglutination is found.
- the reagents which can be used in such immunological test methods can advantageously be packaged in a diagnostic test set for commercial purposes.
- the reagent set for determining an immunologically active substance in a container contains an aqueous suspension of latex particles coated with the corresponding immunological reaction partner.
- the reagent set for determining an immunologically active substance contains a solution of the corresponding immunological reaction partner (e.g. antiserum) in a first container and in. in a second container an aqueous suspension of latex particles coated with the immunologically active material.
- the corresponding immunological reaction partner e.g. antiserum
- the aqueous suspension of the latex-bound immunologically active material or latex-linked immunological reactant can be present in any concentration. However, a concentration of 0.5 to 5% by weight is preferred.
- the polymerization is carried out at 75 ° C. under a nitrogen atmosphere, the monomers being introduced continuously over a period of 7 hours and the reaction being continued for 8 hours.
- Electron microscopy shows that the particles have an average diameter of 0.145 ⁇ m; 90% of the particles have a diameter between 0.14 and 0.15 ⁇ m.
- composition of the polymer is essentially the same as that of the monomers used.
- the particles have carboxyl and sulfonate functions on their surface, which are determined by conductometric titration.
- average diameter of the particles 0.15 m, 90% having a diameter between 0.145 and 0.155 pm.
- the particles have carboxyl and sulfonate functions on their surface, which are confirmed by conductometric titration, and groups of the formula:
- washed latex 1 ml of the 10% latex produced above is added to 20 ml of water and the mixture is centrifuged at 35,000 g for 1 1/2 hours. The supernatant is decanted off, the residue is taken up in 20 ml of water and centrifuged again at 35,000 g for 1 1/2 hours. This operation is repeated twice and the latex thus obtained is referred to as "washed latex" in the following examples.
- the latex is centrifuged off at 35,000 g for 1 1/2 hours, the supernatant is decanted off and the residue is washed twice with 25 ml of 0.1 M glycine-NaOH buffer pH 8.2, by centrifuging and slurrying the residue. So much buffer is now added to the latex that a solution with 30 mg / ml results.
- the following buffer is used for the tube agglutination test: 7.5 g glycine, 6.0 g CaCl 2 , 3 g bovine albumin, 1 g NaN 3 dissolved in 1 liter water. The pH is adjusted to 8.2 with NaOH. To detect the rheumatoid factors in the serum, 20 ⁇ l of latex is diluted with 3 ml of buffer in a small test tube and 25 ⁇ l of the serum to be examined is added. After mixing, the tubes are kept in a heat block at 37 ° C for 2 hours. A positive serum agglutinates under these conditions, while a negative control system shows no agglutination.
- 1 ml of washed latex is prepared and diazotized as in Example 1.
- the diazotized latex residue becomes 5 ml ice-cold 0.1 M glycine-NaOH buffer pH 6.0 was added and 5 mg goat anti-human albumin immunoglobulin G dissolved in 1 ml of the above buffer were added and the mixture was left to stir overnight at 10 ° C. for 1 hour.
- the latex is centrifuged off at 35,000 g for 1 1/2 hours, the supernatant is discarded and the sediment is washed twice with 25 ml of 0.1 M glycine-NaOH pH 8.2. After washing, the latex is mixed with so much buffer that a 3% solution is obtained.
- the following buffer is used for the tube agglutination test: 7.5 g glycine, 6.0 g CaCl 2 , 3 g bovine albumin and 1.0 g NaN 3 are dissolved in 1 liter water and the pH is adjusted to 6.0 with hydrochloric acid. A series of concentrations of human albumin in 3 ml of buffer is prepared in small test tubes, 20 ⁇ l of latex are added in each case and, after mixing, kept in a heat block at 37 ° C. for 2 hours.
- Example 1 1 ml of washed latex from Example 1 is added to 5 ml of 0.1 M phosphate buffer pH 5.0 and 5 mg of sheep anti-human IgG immunoglobulin G in 1 ml of buffer are added and the mixture is stirred well. Then 0.1 ml of a 0.01 M p-phenyldiisothiocyanate solution in dimethylformamide added, stirred for 1 hour and at Allow room temperature to stand overnight. The latex is centrifuged at 35,000 g for 1 1/2 hours, the supernatant is discarded and the residue is washed twice with 25 ml of 0.1 M glycine-NaOH buffer pH 8.2. The latex is used in a concentration of 30 mg / ml for the agglutination test.
- a 0.1 M phosphate buffer pH 6.0 with 0.1% bovine albumin is used for the tube agglutination test.
- a series of concentrations of human IgG in 3 ml of buffer are prepared.
- 20 ⁇ l of latex reagent are added to each tube, mixed and incubated for 2 hours at 37 ° C in a heat block.
- the table shows that 0.1 ⁇ g / ml human IgG can still be determined with this latex reagent.
- Example 1 1 ml of washed latex from Example 1 is taken up in 5 ml of 0.1 M phosphate buffer pH 7.0 and 5 mg of human immunoglobulin G in 1 ml of the above buffer is added. The suspension is cooled to 0 ° and 0.01 ml of a 0.02 M with stirring. bisdiazot convinced benzidine solution was added and then at 1 0 0 left to stand overnight. The latex is centrifuged at 30,000 g for 1 1/2 hours, the supernatant is discarded and the sediment is washed twice with 25 ml of 0.1 M glycine-NaOH pH 8.2. After washing, the latex is mixed so much buffer that a 3% solution is obtained.
- a 0.1 M phosphate buffer pH 6.0 is used to determine IgG in the inhibition test. 3 ml of a 1/500 diluted sheep anti-human IgG serum and increasing amounts of human IgG are placed in small test tubes. After incubation for 15 minutes at 37 ° C, 20 ul latex reagent is added to each tube and incubated for 3 hours at 37 ° C.
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- Polymerisation Methods In General (AREA)
Abstract
immunologisches Reagenz, Verfahren zu deseen Herstellung, Verwendung dieses Reagenzes, Bestimmungsverfahren unter Verwendung dieses Reagenzes und Reagenziengarnitur enthaltend dieses Reagenz.immunological reagent, process for its preparation, use of this reagent, determination method using this reagent and reagent set containing this reagent.
Es wurde ein immunologisches Reagenz mit einem aus Partikeln von Vinylpolymerisaten gebildeten Träger hergestellt, mit dem keine unerwünschte Vernetzung des eingesetzten Antikörpers oder Antigens eintritt. Dieser Träger bildet mit einem breiten Spektrum von immunologiech aktiven Materialien ein diagnostisch verwendbares Reagenz, das Stabil, spezifisch und empfindlich ist Der Träger liegt in Form diskreter Latexteilchen vor; der Latex besteht aus einer Dispersion von ! Partikeln von Vinylpolymerisaten, die als Endgruppen Gruppen der Formel:
Description
Die vorliegende Erfindung betrifft wertvolle diagnostische Reagenzien, ein Verfahren zu deren Herstellung und diagnostische Methoden, worin solche Reagenzien Verwendung finden.The present invention relates to valuable diagnostic reagents, a process for their preparation and diagnostic methods in which such reagents are used.
Die Diagnose von pathologischen oder anderen Zuständen in Menschen und Tieren wird oft unter Anwendung von immunologischen Prinzipien durchgeführt. Diese Prinzipien werden zum Nachweis von Antikörpern oder Antigenen in den Körperflüssigkeiten des Lebewesens benützt. Ein Antigen ist eine fremde Substanz, welche, wenn sie dem Lebewesen appliziert wird, die Bildung von gewissen löslichen und als Antikörper bezeichneten Substanzen bewirkt. Irgendeine Substanz wie z.B. ein Protein, welche normal nicht in einem bestimmten Lebewesen vorhanden ist, kann die Bildung von Antikörpern verursachen, wenn sie dem Lebewesen unter geeigneten Bedingungen appliziert wird.The diagnosis of pathological or other conditions in humans and animals is often carried out using immunological principles. These principles are used to detect antibodies or antigens in the body fluids of the living being. An antigen is a foreign substance which, when applied to the living being, causes the formation of certain soluble substances, known as antibodies. Any substance such as a protein which is not normally present in a particular living being can cause the formation of antibodies if it is applied to the living being under suitable conditions.
Nach ihrer Bildung reagieren die Antikörper mit den Antigenen und schützen auf diese Weise, im Fall eines Bakterien-oder Virus-Fremdkörpers, gegen Infektionen.After their formation, the antibodies react with the antigens and in this way protect against infections in the case of a bacterial or virus foreign body.
Immunologische Testverfahren beruhen auf der Antigen-Antikörper-Reaktion, welche sich gewöhnlich durch Unlöslichkeit oder Agglutination manifestiert.Immunological test methods are based on the antigen-antibody reaction, which usually manifests itself through insolubility or agglutination.
Im allgemeinen wird die Anwesenheit eines Antigens oder eines Antikörpers dadurch bestätigt oder bestimmt, dass man den entsprechenden Antikörper oder das entsprechende Antigen einer Körperflüssigkeit des Lebewesens, meistens Urin, Blutserum oder einem speziell behandelten Blutextrakt, zugibt. Es können jedoch auch andere Körperflüssigkeiten verwendet werden. Man stellt die Anwesenheit bzw. die Abwesenheit des Antikörpers oder des Antigens in der Körperflüssigkeit des Lebewesens fest, indem das Eintreten oder nicht-Eintreten einer Antigen-Antikörperreaktion festgestellt wird.In general, the presence of an antigen or an antibody is confirmed or determined by adding the corresponding antibody or antigen to a body fluid of the living being, usually urine, blood serum or a specially treated blood extract. However, other body fluids can also be used. The presence or absence of the antibody or the antigen in the body fluid of the living being is determined by determining the occurrence or non-occurrence of an antigen-antibody reaction.
Weil einige Komplexe sich nur sehr langsam bilden und sehr geringe Teilchengrössen besitzen, ist es notwendig,Träger zu benützen, um sie sichtbar zu machen. In einer bisher bevorzugten Methode wurden der Antikörper bzw. das Antigen mittels' eines Carbodiimids über eine Amidbindung an diskreten Teilchen von carboxylierten Latexpolymeren,wie z.B. von carboxylierten Copolymeren aus Butadien und Styrol,gebunden.Because some complexes form very slowly and have very small particle sizes, it is necessary to use carriers to make them visible. In a previously preferred method, the antibody or antigen was' by means of a carbodiimide via an amide bond to discrete particles of carboxylated latex polymers, such as e.g. of carboxylated copolymers of butadiene and styrene.
Diese Methode hat jedoch den Nachteil, dass während der Kupplung des Proteins (Antikörper oder Antigen) an die Latexteilchen, wegen der Verwendung von Carbodiimiden als Nebenreaktion eine unerwünschte Vernetzung des eingesetzten Proteins eintritt und somit ein Teil der oft sehr teuren Proteine für die Kupplung mit dem Träger verloren geht.However, this method has the disadvantage that during the coupling of the protein (antibody or antigen) to the latex particles, because of the use of carbodiimides as a side reaction, undesired crosslinking of the protein used occurs and thus part of the often very expensive proteins for coupling with the Carrier is lost.
Die vorliegende Erfindung betrifft nun neuartige polymere Träger, mit welchen die obigen Nachteile vermieden werden können und welche mit einem breiten Spektrum von immunologisch aktiven Materialien ein diagnostisch verwendbares Reagenz bilden können, das stabil, spezifisch und empfindlich ist und eine leicht nachweisbare visuelle Bewertung in sehr kurzer Zeit ermöglicht.The present invention now relates to novel polymeric carriers with which the above disadvantages can be avoided and which with a wide range of immunologically active Materials can form a diagnostically usable reagent that is stable, specific, and sensitive, and that enables an easily detectable visual assessment in a very short time.
Genauer gesagt betrifft die vorliegende Erfindung ein wasserunlösliches Reagenz für eine immunologische Bestimmung mit einem etwa dem von Wasser entsprechenden spezifischen Gewicht in Form diskreter Latexteilchen, an welche ein immunologisch aktives Material gebunden ist, dadurch gekennzeichnet, dass der Latex aus einer Dispersion von Partikeln von Vinylpolymerisaten besteht, die als Endgruppen Gruppen der Formel:
Weiter bezieht sich die Erfindung auf ein Verfahren zur Herstellung eines solchen Reagens, welches dadurch gekennzeichnet ist, dass man den Latex nach Diazotierung oder in Gegenwart von geeigneten bifunktionellen Reagenzien mit dem immunologisch aktiven Material umsetzt.The invention further relates to a method for producing such a reagent, which is characterized in that the latex is reacted with the immunologically active material after diazotization or in the presence of suitable bifunctional reagents.
Als "immunologisch aktive Substanzen" können all jene Bestandteile in physiologischen Flüssigkeiten, Zell- und Gewebeextrakten genannt werden, für die ein immunologischer Reaktionspartner vorhanden ist oder gebildet werden kann. Dazu gehören Amine, Aminosäuren, Peptide, Proteine, Lipoproteine, Glycoproteide, Sterine, Steroide, Lipoide, Nucleinsäuren, Enzyme, Hormone, Vitamine, Polysaccharide und Alkaloide. Bevorzugte immunologisch aktive Substanzen sind in der folgenden Tabelle zusammengestellt:
Im Rahmen der vorliegenden Erfindung sind besonders bevorzugte immunologisch aktive Substanzen Albumin, Rheumatoidfaktor, menschliches Immunoglobulin IgG und Antikörper gegen IgG.In the context of the present invention, particularly preferred immunologically active substances are albumin, rheumatoid factor, human immunoglobulin IgG and antibodies against IgG.
Unter Vinylpolymerisaten, die den Kern der Partikeln bilden, versteht man Homopolymerisate von Monomeren, wie Styrol und seine Derivate: Methylstyrole, Aethylstyrole, Vinyltoluol; Vinylchlorid, Vinylidenchlorid; Vinylacetat; Acrylderivate, wie Alkylacrylate und -methacrylate (Alkyl mit 1 bis 10 Kohlenstoffatomen), die gegebenenfalls hydroxyliert sind, wie 2-Hydroxyäthylacrylat und -methacrylat und 2-Hydroxypropylacrylat und -methacrylat; Acrylnitril und Methacrylnitril; sowie Copolymerisate dieser Monomeren untereinander und/oder mit modifizierenden Vinylcomonomeren, wie Divinylbenzol, Acrylamid und Methacrylamid und deren N-substituierte Derivate, wie z.B. Methylolacrylamid; diese Comonomere stellen bis zu 5 Gew.-% des Copolymerisates dar.Vinyl polymers which form the core of the particles are understood to mean homopolymers of monomers, such as styrene and its derivatives: methylstyrenes, ethylstyrenes, vinyltoluene; Vinyl chloride, vinylidene chloride; Vinyl acetate; Acrylic derivatives, such as alkyl acrylates and methacrylates (alkyl having 1 to 10 carbon atoms) which are optionally hydroxylated, such as 2-hydroxyethyl acrylate and methacrylate and 2-hydroxypropyl acrylate and methacrylate; Acrylonitrile and methacrylonitrile; and copolymers of these monomers with one another and / or with modifying vinyl comonomers such as divinylbenzene, acrylamide and methacrylamide and their N-substituted derivatives, such as e.g. Methylolacrylamide; these comonomers represent up to 5% by weight of the copolymer.
Unter Dienpolymerisaten, die den Kern bilden, versteht man Homopolymerisate des Butadiens und seiner Derivate: Chloropren, Isopren; sowie die Copolymerisate dieser Monomeren untereinander und/oder mit Vinylmonomeren, wie sie oben genannt wurden, in allen Mengenverhältnissen und/oder mit modifizierend wirkenden Vinylmonomeren, wie sie oben'aufgezählt wurden, deren Menge in dem Copolymerisat bis zu 5 Gew.-% ausmacht.Diene polymers that form the core are homopolymers of butadiene and its derivatives: chloroprene, isoprene; and the copolymers of these monomers with one another and / or with vinyl monomers, as mentioned above, in all proportions and / or with modifying vinyl monomers, as enumerated above, the amount of which in the copolymer makes up to 5% by weight.
Die Vinylpolymerisate, welche die äussere Schicht der Partikel bilden, sind Homopolymerisate von Monomeren, wie Styrol und seine Derivate, z.B. Methylstyrole, Aethylstyrole und Vinyltoluol; gegebenenfalls hydroxylierte Alkylacrylate und Alkylmethacrylate (Alkyl mit 1 bis 10 Kohlenstoffatomen); Acrylnitril und Methacrylnitril; sowie Copolymerisate dieser Monomeren untereinander und/oder mit modifizierend wirkenden Vinylcomonomeren, wie Divinylbenzol, Acrylamid und Methacrylamid sowie deren N-substituierte Derivate, wie Methylolacrylamid, die bis zu 5 Gew.-% des Copolymerisats ausmachen können.The vinyl polymers which form the outer layer of the particles are homopolymers of monomers, such as styrene and its derivatives, for example methylstyrenes, ethylstyrenes and vinyl toluene; optionally hydroxylated alkyl acrylates and alkyl methacrylates (alkyl with 1 to 10 carbon atoms); Acrylonitrile and methacrylonitrile; and copolymers of these monomers with one another and / or with modifying vinyl comonomers, such as divinylbenzene, acrylamide and methacrylamide, and their N-substituted derivatives, such as methylolacrylamide, which can make up up to 5% by weight of the copolymer.
In den Partikeln stellt das Kernpolymerisat 30 bis 99,5 Gew.-%, vorzugsweise 60 bis 99 Gew.-%, und das Polymerisat der äusseren Schicht 70 bis 0,5 Gew.-%, vorzugsweise 40 bis 1 Gew.-%, dar.The core polymer in the particles constitutes 30 to 99.5% by weight, preferably 60 to 99% by weight, and the polymer in the outer layer 70 to 0.5% by weight, preferably 40 to 1% by weight, represents.
Die Polymerisatpartikel, deren Kornverteilung je nach den gewünschten Eigenschaften des Latex und den in Betracht gezogenen Anwendungen breit oder eng sein kann, haben einen mittleren Durchmesser zwischen 0,03 und 5 µm, vorzugsweise zwischen 0,05 und 1 ym. Sie stellen bis zu 60 Gew.-%, vorzugsweise bis zu 45 Gew.-%, des Latex dar. Jedoch kann der Latex ohne weiteres verdünnt oder konzentriert werden.The polymer particles, the particle size distribution of which may be wide or narrow depending on the desired properties of the latex and the applications under consideration, have an average diameter between 0.03 and 5 μm, preferably between 0.05 and 1 μm. They represent up to 60% by weight, preferably up to 45% by weight, of the latex. However, the latex can easily be diluted or concentrated.
Das Kernpolymerisat kann hergestellt werden durch Emulsionspolymerisation des oder der Vinylmonomeren und/oder Dienmonomeren in Gegenwart von mindestens einer äthylenischen Mono-oder Polycarbonsäure, die mit dem oder den Monomeren copolymerisierbar ist, und/oder mindestens eines copolymerisierbaren ungesättigten Alkaliorganosulfonats; dann wird das Polymerisat der äusseren Schicht hergestellt durch Emulsionspolymerisation des oder der Vinylmonomeren in Gegenwart des Latex des Kernpolymerisates, der oben erhalten wurde, und in Gegenwart eines Kettenübertragungsmittels.The core polymer can be prepared by emulsion polymerization of the vinyl monomer (s) and / or diene monomer (s) in the presence of at least one ethylenic mono- or polycarboxylic acid which is copolymerizable with the monomer (s) and / or at least one copolymerizable unsaturated alkali metal organosulfonate; then the polymer of the outer layer is prepared by emulsion polymerizing the vinyl monomer (s) in the presence of the latex of the core polymer obtained above and in the presence of a chain transfer agent.
Die bei der Polymerisation des Kernpolymerisates und bei der Polymerisation des Polymerisates der äusseren Schicht verwendeten Monomeren sind die oben aufgezählten Monomeren. Sie werden entweder alle vor der Polymerisation verwendet oder zum Teil vor der Polymerisation verwendet, wobei der restliche Teil im Verlauf der Polymerisation in aufeinanderfolgenden Fraktionen oder kontinuierlich zum Reaktionsmedium gegeben wird, oder alle im Verlauf der Polymerisation in aufeinanderfolgenden Fraktionen oder kontinuierlich zugesetzt.The monomers used in the polymerization of the core polymer and in the polymerization of the polymer of the outer layer are the monomers listed above. They are either all used before the polymerization or for Part used before the polymerization, the remaining part being added to the reaction medium in successive fractions or continuously in the course of the polymerization, or all added in successive fractions or continuously in the course of the polymerization.
Als copolymerisierbare äthylenische Mono- oder Polycarbonsäuren seien Acrylsäure, Methacrylsäure, Maleinsäure, Fumarsäure, Crotonsäure, Sorbinsäure, Zimtsäure, Itaconsäure, Aconitsäure genannt, die in Mengen zwischen 0,5 und 15 Gew.-%, vorzugsweise zwischen 0,5 und 10 Gew.-%, bezogen auf das oder die Monomeren, verwendet werden.As copolymerizable ethylenic mono- or polycarboxylic acids, acrylic acid, methacrylic acid, maleic acid, fumaric acid, crotonic acid, sorbic acid, cinnamic acid, itaconic acid, aconitic acid may be mentioned, in amounts between 0.5 and 15% by weight, preferably between 0.5 and 10% by weight. -%, based on the monomer or monomers used.
Die copolymerisierbaren ungesättigten Alkaliorganosulfonate sind z.B. Natriumvinylsulfonat, Natriummethallylsulfonat, Natrium-2-sulfoäthylacrylat, Natrium-2-sulfoäthylmetacrylat, 2-Acrylamido-2-methylpropansulfonat; sie werden in Mengen zwischen 0,1 und 3 Gew.-%, bezogen auf das oder die Monomeren, verwendet.The copolymerizable unsaturated alkali organosulfonates are e.g. Sodium vinyl sulfonate, sodium methallyl sulfonate, sodium 2-sulfoethyl acrylate, sodium 2-sulfoethyl methacrylate, 2-acrylamido-2-methylpropane sulfonate; they are used in amounts between 0.1 and 3% by weight, based on the monomer or monomers.
Die copolymerisierbaren äthylenischen Mono- oder Polycarbonsäuren und die copolymerisierbaren ungesättigten Alkaliorganosulfonate können einzeln oder in Kombination in den angegebenen Mengen verwendet werden.The copolymerizable ethylene mono- or polycarboxylic acids and the copolymerizable unsaturated alkali organosulfonates can be used individually or in combination in the amounts specified.
Die Herstellung des Kernpolymerisates erfolgt in Emulsion nach jedem beliebigen klassischen Verfahren in Gegenwart eines Initiators und eines Emulgators.The core polymer is prepared in emulsion by any conventional method in the presence of an initiator and an emulsifier.
Als Initiator verwendet man vorzugsweise Alkalipersulfate, wasserlösliche Diazoderivate oder Redoxysysteme auf Basis von Wasserstoffperoxyd, organischen Peroxyden oder Hydroperoxyden in Mengen in der Grössenordnung von 0,01 bis 5 Gew.-%, vorzugsweise 0,03 bis 3 Gew.-%, bezogen auf das oder die Monomeren.The initiator used is preferably alkali metal sulfates, water-soluble diazo derivatives or redox systems based on hydrogen peroxide, organic peroxides or hydroperoxides in amounts of the order of 0.01 to 5% by weight, preferably 0.03 to 3% by weight, based on the or the monomers.
Der verwendete Emulgator kann anionaktiv und/oder nichtionogen sein. Es handelt sich um klassische Produkte für die Emulsionspolymerisation.The emulsifier used can be anionic and / or nonionic. These are classic products for emulsion polymerization.
Als anionaktive Emulgatoren seien genannt Salze von Fettsäuren; Alkalialkylsulfate, Alkalialkylsulfonate, Alkalialkylarylsulfonate, Alkalialkylsulfosuccinate, Alkalialkylphosphate; Sulfobernsteinsäurealkylester; Sulfonate von Alkylphenolpolyglycoläthern; Salze von Estern von Alkylsulfopolycarbonsäuren; Kondensationsprodukte von Fettsäuren mit Oxyalkansulfonsäuren und Aminoalkansulfonsäuren; sulfatierte Derivate von Pölyglycoläthern; sulfatierte Ester von Fettsäuren und Polyglycolen; Alkanolamide von sulfatierten Fettsäuren.Salts of fatty acids may be mentioned as anionic emulsifiers; Alkaline alkyl sulfates, Alkalialkylsulfonate, Alkalialkylarylsulfonate, Alkalialkylsulfosuccinate, Alkalialkylphosphate; Alkyl sulfosuccinate; Sulfonates of alkylphenol polyglycol ethers; Salts of esters of alkylsulfopolycarboxylic acids; Condensation products of fatty acids with oxyalkanesulfonic acids and aminoalkanesulfonic acids; sulfated derivatives of polyglycol ethers; sulfated esters of fatty acids and polyglycols; Alkanolamides of sulfated fatty acids.
Als nichtionogene Emulgatoren kommen Fettsäureester von Polyalkoholen, Alkanolamide von Fettsäuren, Polyäthylenoxyde, Copolyäthylenoxyd/Propylenoxyd, oxyäthylierte Alkylphenole in Betracht.Suitable nonionic emulsifiers are fatty acid esters of polyalcohols, alkanolamides of fatty acids, polyethylene oxides, copolyethylene oxide / propylene oxide and oxyethylated alkylphenols.
Die zu verwendenden Mengen des oder der Emulgatoren liegen in der Grössenordnung von 0,01 bis 5 Gew.-%, bezogen auf das oder die Monomeren, und ihre Einführung erfolgt entweder insgesamt vor der Polymerisation oder zum Teil vor der Polymerisation, wobei der restliche Teil im Verlauf der Polymerisation in nacheinanderfolgenden Fraktionen oder kontinuierlich zum Reaktionsmedium zugesetzt wird, oder insgesamt im Verlauf der Polymerisation in nacheinanderfolgenden Fraktionen oder kontinuierlich.The amounts of the emulsifier or emulsifiers to be used are of the order of magnitude of 0.01 to 5% by weight, based on the monomer or monomers, and they are introduced either in total before the polymerization or in part before the polymerization, the remaining part is added in the course of the polymerization in successive fractions or continuously to the reaction medium, or overall in the course of the polymerization in successive fractions or continuously.
Die für die Polymerisation des Kernpolymerisates zu verwendende Menge Wasser muss derart sein, dass die Konzentration des oder der Monomeren 60 Gew.-% nicht übersteigt.The amount of water to be used for the polymerization of the core polymer must be such that the concentration of the monomer or monomers does not exceed 60% by weight.
Obgleich es nicht unbedingt erforderlich ist, ist es möglich, dem Reaktionsmedium beliebige Verbindungen zuzusetzen, die entweder die Ionenstärke des Mediums und demzufolge die Kornverteilung zu modifizieren vermögen, wie Mineralsalze, Elektrolyten, in einer Menge bis zu 3 Gew.-%, bezogen auf die Monomeren, oder die den pH-Wert des Mediums zu modifizieren vermögen, wie beispielsweise Puffer, Säuren, Basen. Jedoch wurde in bestimmten Fällen festgestellt, dass es zur Begünstigung der Copolymerisation zu bevorzugen ist, wenn das Medium neutral oder sauer ist.Although it is not absolutely necessary, it is possible to add any compounds to the reaction medium which are capable of modifying either the ionic strength of the medium and consequently the particle size distribution, such as mineral salts, electrolytes, in an amount of up to 3% by weight, based on the Monomers, or which can modify the pH of the medium, such as buffers, acids, bases. However it has been found in certain cases that it is preferable to favor the copolymerization if the medium is neutral or acidic.
Die Polymerisationstemperatur, die eine Funktion des verwendeten Initiators und des herzustellenden Polymerisats ist, liegt im allgemeinen zwischen -5 und +90°C.The polymerization temperature, which is a function of the initiator used and the polymer to be prepared, is generally between -5 and + 90 ° C.
Die erhaltenen Latices weisen Polymerisatpartikel mit einem Durchmesser zwischen 0,03 und 5 µm, vorzugsweise zwischen 0,05 und 1 ktm, auf. Diese Partikel sind im allgemeinen nicht kalibriert, aber es ist möglich, sie kalibriert zu erhalten, wenn man bekannte Kalibrierverfahren für die Emulsionspolymerisation anwendet, wie die gesteuerte Zugabe des Emulgators und/oder des oder der Monomeren und insbesondere die Animpfung. Im letzten Falle kann der Emulgator in dem Impfmaterial enthalten sein.The latices obtained have polymer particles with a diameter between 0.03 and 5 μm, preferably between 0.05 and 1 ktm. These particles are generally not calibrated, but it is possible to obtain them calibrated using known calibration methods for emulsion polymerization, such as the controlled addition of the emulsifier and / or the monomer (s) and in particular the inoculation. In the latter case, the emulsifier can be contained in the inoculum.
Die Partikel sind aus Homopolymerisat oder Copolymerisat mit einer Oberfläche von Carboxyl- und/oder Sulfonatfunktionen gebildet. Das Vorhandensein dieser Funktionen kann durch konduktometrische Titration bestätigt werden.The particles are formed from homopolymer or copolymer with a surface of carboxyl and / or sulfonate functions. The presence of these functions can be confirmed by conductometric titration.
Die Herstellung des Polymerisates der äusseren Schicht wird in wässriger Emulsion in Gegenwart von Kernpolymerisat, Kettenübertragungsmittel, Initiator und gegebenenfalls Emulgator ausgeführt.The preparation of the polymer of the outer layer is carried out in an aqueous emulsion in the presence of the core polymer, chain transfer agent, initiator and, if appropriate, emulsifier.
Die verwendete Menge des Kernpolymerisats liegt zwischen 30 und 99,5 Gew.-% und beträgt.vorzugsweise 60 bis 99 Gew.-%, bezogen auf die Summe von Kernpolymerisat und zu polymerisierendem Monomer oder zu polymerisierenden Monomeren.The amount of the core polymer used is between 30 and 99.5% by weight and is preferably 60 to 99% by weight, based on the sum of the core polymer and the monomer or monomers to be polymerized.
Das Kettenübertragungsmittel vom Typ Aminophenyldisulfid oder Aminophenylmercaptan ist insbesondere o,o'-Dithiobisani- lin, p,p'-Dithiobisanilin, 2-Mercaptoanilin, 3-Mercaptoanilin, 4-Mercaptoanilin. Dieses Mittel wird im allgemeinen in Lösung in dem oder den Monomeren verwendet, und zwar in Mengen zwischen O,1 und 10 Gew.-%, vorzugsweise zwischen 0,5 und 5 Gew.-%, bezogen auf das oder die Monomere.The chain transfer agent of the aminophenyl disulfide or aminophenyl mercaptan type is in particular o, o'-dithiobisaniline, p, p'-dithiobisaniline, 2-mercaptoaniline, 3-mercaptoaniline, 4-mercaptoaniline. This remedy is generally in solution used in the monomer or monomers, in amounts between 0.1 and 10% by weight, preferably between 0.5 and 5% by weight, based on the monomer or monomers.
Die für die Polymerisation des oder der Monomeren der äusseren Schicht erforderlichen Initiatoren sind Diazoinitiatoren, Azonitrile wie Azo-bis-isobutyronitril oder wie sulfonierte Azonitrile, wie sie im französischen Patent Nr. 1.233.582 beschrieben sind; von diesen kann man erwähnen Azobis-(iso- butyronitrilnatriumsulfonat), Azobis-(a-methylbutyronitril- natriumsulfonat), Azobis-(a-methyl-ß-äthoxycarbonylbutyronitril- natriumsulfonat); carboxylierte Azonitrile, wie 4,4'-Azobis-(4- cyanpentansäure) und ihre Salze, Azobis-alkylamidiniumsalze, wie a,a'-Azobis-isobutyramidiniumchlorid, Azobis-N,N'-dimethylen- isobutyramidiniumchlorid.The initiators required for the polymerization of the outer layer or monomers are diazo initiators, azonitriles such as azo-bis-isobutyronitrile or such as sulfonated azonitriles as described in French Patent No. 1,233,582; of these, one can mention azobis (isobutyronitrile sodium sulfonate), azobis (a-methylbutyronitrile sodium sulfonate), azobis (a-methyl-β-ethoxycarbonylbutyronitrile sodium sulfonate); carboxylated azonitriles such as 4,4'-azobis (4-cyanopentanoic acid) and their salts, azobis-alkylamidinium salts such as a, a'-azobis-isobutyramidinium chloride, azobis-N, N'-dimethylene-isobutyramidinium chloride.
Der Initiator, der in einer Menge von 0,01 bis 3 Gew.-%, vorzugsweise 0,1 bis 2 Gew.-%, bezogen auf das oder die Monomeren, verwendet wird, wird insgesamt oder zum Teil vor der Polymerisation verwendet, wobei der andere Teil im Verlauf der Polymerisation in nacheinanderfolgenden Fraktionen oder kontinuierlich zu dem Reaktionsmedium zugesetzt wird, insbesondere wenn die Lebensdauer des Initiators bei der Polymerisationstemperatur kurz ist. Der Initiator kann auch insgesamt im Verlauf der Polymerisation kontinuierlich zum Reaktionsmedium zugegeben werden.The initiator, which is used in an amount of 0.01 to 3% by weight, preferably 0.1 to 2% by weight, based on the monomer or monomers, is used in whole or in part before the polymerization, where the other part is added to the reaction medium in successive fractions or continuously in the course of the polymerization, in particular if the life of the initiator at the polymerization temperature is short. The initiator can also be added continuously to the reaction medium overall in the course of the polymerization.
Der allfällige Emulgator wird aus anionaktiven und/oder nichtionogenen Emulgatoren gewählt, die für die Herstellung des Kernpolymerisats angegeben wurden; er kann gleich oder verschieden wie der für die Herstellung des Kernpolymerisates verwendete Emulgator sein. Er wird in Mengen von bis zu 10 Gew.-%, bezogen auf das oder die Monomeren, verwendet, und seine Einführung kann je nach dem mittleren Durchmesser der Latexpartikel, der erhalten werden soll, entweder insgesamt vor der Polymerisation oder zum Teil vor der Polymerisation erfolgen, wobei der restliche Teil im Verlauf der Polymerisation in nacheinanderfolgenden Fraktionen oder kontinuierlich zugegeben wird, oder sie kann insgesamt im Verlauf der Polymerisation in aufeinanderfolgenden Fraktionen oder kontinuierlich erfolgen.The emulsifier, if any, is selected from anionic and / or nonionic emulsifiers which have been specified for the preparation of the core polymer; it can be the same or different from the emulsifier used for the production of the core polymer. It is used in amounts of up to 10% by weight, based on the monomer or monomers, and depending on the average diameter of the latex particles to be obtained, it can be introduced either entirely before the polymerization or partly before the polymerization take place, the remaining part in the course of the polymer tion is added in successive fractions or continuously, or it can be done in the course of the polymerization in successive fractions or continuously.
Die bei der Polymerisation der äusseren Schicht zu verwendende Menge Wasser muss derart sein, dass die Konzentration von Kernpolymerisat und zu polymerisierendem oder zu polymerisierenden Monomeren 60 Gew.-%, vorzugsweise 45 Gew.-%, nicht übersteigt.The amount of water to be used in the polymerization of the outer layer must be such that the concentration of core polymer and monomers to be polymerized or polymerized does not exceed 60% by weight, preferably 45% by weight.
Die Polymerisationstemperatur, die eine Funktion des gewählten Initiators ist, liegt im allgemeinen zwischen 5 und 100°C, vorzugsweise zwischen 40 und 90°C.The polymerization temperature, which is a function of the chosen initiator, is generally between 5 and 100 ° C, preferably between 40 and 90 ° C.
Die erhaltenen Latices weisen Polymerisationspartikel auf, deren Durchmesser zwischen 0,03 und 5 µm, vorzugsweise zwischen 0,05 und 1 µm, liegt; da die Menge der äusseren Schicht nicht sehr gross ist, modifiziert sie nicht in merklicher Weise die Grösse der Partikel des Kernpolymerisates. Die Partikel können kalibriert sein oder nicht, aber bei bestimmten Anwendungen wird es aus Gründen der Reproduzierbarkeit bevorzugt, dass sie kalibriert sind, d.h. dass sie eine schmale Korngrössenverteilung haben.The latices obtained have polymerization particles whose diameter is between 0.03 and 5 μm, preferably between 0.05 and 1 μm; since the amount of the outer layer is not very large, it does not noticeably modify the size of the particles of the core polymer. The particles may or may not be calibrated, but in certain applications it is preferred for reasons of reproducibility that they are calibrated, i.e. that they have a narrow grain size distribution.
Die Latices sind mechanisch und bei der Lagerung sowie gegen Elektrolyten beständig, d.h. sie flocken nicht aus, wenn man ihnen Mineralsalze, wie z.B. die Chloride, Nitrate, Borate, Phosphate des Natriums, Calciums, Magnesiums, Kaliums zusetzt.The latices are mechanical and resistant to storage and electrolytes, i.e. they do not flocculate if you are given mineral salts such as the chlorides, nitrates, borates, phosphates of sodium, calcium, magnesium, potassium are added.
Die Partikel sind aus Polymerisaten gebildet und weisen eine Oberfläche mit Carboxyl- und/oder Sulfonatfunktionen sowie Gruppen der Formel:
Obgleich die äussere Schicht auf dem Kernpolymerisat polymerisiert wird, bleiben die Carboxyl- und/oder Sulfonatfunktionen zugänglich, wie durch konduktometrische Titration gezeigt werden kann, und die Gruppen der Formel:
Die immunologisch aktiven Materialien (Antigen oder Antikörper) können physikalisch und/oder chemisch an den erfindungsgemäss verwendeten Latexpolymeren gebunden werden.The immunologically active materials (antigen or antibody) can be bound physically and / or chemically to the latex polymers used according to the invention.
In einer Ausführungsform wird das erfindungsgemässe Reagenz durch Bildung einer Azobindung zwischen dem Latex und dem immunologisch aktiven Material hergestellt. Zu diesem Zweck werden durch Behandlung des Latex in wässriger saurer Lösung mit einem Nitrit die primären aromatischen Aminogruppen des Latex in ein Diazoniumsalz überführt.In one embodiment, the reagent according to the invention is produced by forming an azo bond between the latex and the immunologically active material. For this purpose, by treating the latex in aqueous acid solution with a nitrite, the primary aromatic amino groups of the latex are converted into a diazonium salt.
Als Säure kann z.B. eine anorganische Säure wie Salzsäure, Schwefelsäure oder Perchlorsäure verwendet werden. Als Nitrit wird vorzugsweise Natriumnitrit oder Kaliumnitrit verwendet. Die Reaktion wird wegen der Instabilität der Diazoniumsalze vorzugsweise bei 0-5 C ausgeführt.As the acid e.g. an inorganic acid such as hydrochloric acid, sulfuric acid or perchloric acid can be used. Sodium nitrite or potassium nitrite is preferably used as the nitrite. The reaction is preferably carried out at 0-5 C because of the instability of the diazonium salts.
Das immunologische aktive Material wird anschliesscnd im wässrigen Medium, vorzugsweise zwischen 0°C und Zimmertemperatur,mit dem diazotierten Träger zur Reaktion gebracht.The immunologically active material is then reacted with the diazotized carrier in an aqueous medium, preferably between 0.degree. C. and room temperature.
In einer weiteren Ausführungsform des erfindungsgemässen Verfahrens kann das immunologisch aktive Material mit Hilfe einer polyfunktionellen Verbindung über ein Zwischenstück an den gemäss vorliegender Erfindung verwendeten Latex gebunden werden.In a further embodiment of the method according to the invention, the immunologically active material can be bound to the latex used according to the present invention by means of a polyfunctional compound via an intermediate piece.
Als polyfunktionelle Verbindungen eignen sich diejenigen, welche mit den aromatischen Aminogruppen des Latexpolymeren reagieren oder an den aromatischen Ring des Latexpolymeren eine Substitutionsreaktion eingehen und gleichzeitig mit funktionellen Gruppen des immunologisch aktiven Materials, wie Amino-, Mercapto-, Carbonyl- and Hydroxylgruppen, reagieren oder eine Substitutionsreaktion an dem aromatischen Ring des immunologisch aktiven Materials eingehen.As polyfunctional compounds are useful ones which react with the aromatic amino groups of the latex polymer or undergo a substitution reaction at the aromatic ring of the latex polymer and at the same mino- with functional groups of the immunologically active material such as A, mercapto, carbonyl and hydroxyl groups react or undergo a substitution reaction on the aromatic ring of the immunologically active material.
Repräsentative Vertreter derartiger polyfunktioneller Verbindungen sind Azó-, Isocyano-, Isothiocyano- oder Aldehydgruppen enthaltenden Verbindungen wie z.B. Bis-diazobenzidin, Bis-diazobenzidin-disulfonsäure, Bis-diazo-p-phenyldiamin, Phenyldiisocyanat, Toluoldiisocyanat, Glutardialdehyd.Representative representatives of such polyfunctional compounds are azó, isocyano, isothiocyano or aldehyde group-containing compounds such as e.g. Bis-diazobenzidine, bis-diazobenzidine-disulfonic acid, bis-diazo-p-phenyl diamine, phenyl diisocyanate, toluene diisocyanate, glutardialdehyde.
Bei Umsetzung in Gegenwart einer bifunktionellen Verbindung wird das immunologisch aktive Material in wässrigem Medium, vorzugsweise bei Zimmertemperatur (20°C bis 25°C), mit dem Träger zur Reaktion gebracht. Die Temperatur kann jedoch auch zwischen 0°C und 40°C liegen.When reacting in the presence of a bifunctional compound, the immunologically active material is reacted with the carrier in an aqueous medium, preferably at room temperature (20 ° C. to 25 ° C.). However, the temperature can also be between 0 ° C and 40 ° C.
Die Menge der verwendeten bifunktionellen Verbindung hängt ab von der Zahl der Aminogruppen auf dem Latex. Vorzugsweise wird ein zehn- bis hundert-facher molarer Ueberschuss der bifunktionellen Verbindung gegenüber der Zahl der Aminogruppen des eingesetztes Latex verwendet.The amount of the bifunctional compound used depends on the number of amino groups on the latex. A ten- to hundred-fold molar excess of the bifunctional compound compared to the number of amino groups of the latex used is preferably used.
In beiden Ausführungsformen zur Herstellung des erfindungsgemässen Reagenz ist der pH-Wert der Reaktion wichtig. Er darf nicht so gewählt werden, dass ein Protein-Reaktionspartner denaturiert wird. In der Regel liegt der pH-Wert zwischen 5 und 9. Dieser pH-Wert wird unter Verwendung von geeigneten üblichen. Puffer-Systemen Wie Phosphatpuffer und dgl., aufrechterhalten.In both embodiments for the preparation of the reagent according to the invention, the pH of the reaction is important. It should not be chosen to denature a protein reactant. Usually the pH is between 5 and 9. This pH is determined using suitable ones. Buffer systems such as phosphate buffers and the like are maintained.
Das Endprodukt ist ein wassernlösliches Material, welches in einer wässrigen Pufferlösung von pH-Wert 5,0 bis 9 suspendiert ist, wobei der pH-Wert der Lösung von dem im einzelnen verwendeten System und von den Anforderungen an die Stabilität des immunologisch aktiven Materials abhängig ist. Das spezifische Gewicht des Produktes entspricht etwa demjenigen von Wasser (0,97-1,02), wodurch eine stabile Suspension des Produkts erreicht wird. Die Produkte können z.B. durch Zentrifugieren in Form eines weissen oder gelblichen Niederschlags isoliert werden.The final product is a water-soluble material which is suspended in an aqueous buffer solution of pH 5.0 to 9, the pH of the solution being different from that in detail used system and depends on the requirements for the stability of the immunologically active material. The specific weight of the product corresponds approximately to that of water (0.97-1.02), whereby a stable suspension of the product is achieved. The products can be isolated, for example, by centrifugation in the form of a white or yellowish precipitate.
Die Menge an immunologisch aktivem Material, das an die immunologisch inerten LatexpolymernTräger gebunden ist, beträgt in der Regel 0,01 bis 15,0 Gew.%. Jedoch wird jedes einzelne immunologisch aktive Material in einer Menge benützt, welche sich in einem diagnostischen Test am zweckmässigsten erweist. Aus diesem Grunde wird jedes Material mit dem Träger in einem Verhältnis kombiniert, welches den jeweiligen spezifischen Anforderungen am besten entspricht. Die vorliegende Erfindung umfasst deshalb die Verwendung einer solchen Menge an immunologisch aktivem Material in Kombination mit einem immunologisch inerten Latexpolymer-Träger, die geeignet ist, ein für derartige diagnostische Zwecke nützliches Reagens zu liefern.The amount of immunologically active material which is bound to the immunologically inert latex polymer carrier is usually 0.01 to 15.0% by weight. However, each individual immunologically active material is used in an amount which is most useful in a diagnostic test. For this reason, each material is combined with the carrier in a ratio that best suits the specific requirements. The present invention therefore encompasses the use of such an amount of immunologically active material in combination with an immunologically inert latex polymer carrier which is suitable for providing a reagent useful for such diagnostic purposes.
Nach seiner Herstellung kann das Produkt in spezifischen diagnostischen Tests, welche auf immunologischen Prinzipien aufgebaut sind, verwendet werden.Once manufactured, the product can be used in specific diagnostic tests based on immunological principles.
Erfindungsgemäss kann die Bestimmung der immunologisch aktiven Substanz sowohl in einem direkten wie auch in einem indirekten (Inhibitions-) Testverfahren durchgeführt werden.According to the invention, the determination of the immunologically active substance can be carried out both in a direct and in an indirect (inhibition) test method.
Im direkten Testverfahren werden zur Bestimmung einer immunologisch aktiven Substanz die Analysenprobe und die mit dem entsprechenden immunologischen Reaktionspartner beschichteten Latexteilchen vermischt und das Auftreten einer Agglutination beobachtet. Der Test ist positiv, wenn eine Agglutination festgestellt wird.In the direct test method, the analytical sample and the latex particles coated with the corresponding immunological reaction partner are mixed to determine an immunologically active substance and the occurrence of agglutination is observed. The test is positive if agglutination is detected.
Beim indirekten (Inhibitions-)-Testverfahren wird zur Bestimmung einer immunologisch aktiven Substanz die Analysenprobe mit einer bestimmten Menge des entsprechenden immunologischen Reaktionspartners(z.B. Antiserum) und Latexteilchen, die mit der immunologisch aktiven Substanz beschichtet sind, vermischt und das Auftreten einer Agglutination beobachtet. Der Test ist positiv, wenn keine Agglutination festgestellt wird.In the indirect (inhibition) test method, the analytical sample is mixed with a certain amount of the corresponding immunological reaction partner (e.g. antiserum) and latex particles coated with the immunologically active substance to determine an immunologically active substance and the occurrence of agglutination is observed. The test is positive if no agglutination is found.
Die in derartigen immunologischen Testverfahren nutzbaren Reagenzien können vorteilhaft für kommerzielle Zwecke in eine diagnostische Testgarnitur abgepackt werden.The reagents which can be used in such immunological test methods can advantageously be packaged in a diagnostic test set for commercial purposes.
Im Falle eines direkten Tests enthält die Reagenziengarnitur zur Bestimmung einer immunologisch aktiven Substanz in einem Behälter eine wässrige Suspension von mit dem entsprechenden immunologischen Reaktionspartner beschichteten Latexteilchen.In the case of a direct test, the reagent set for determining an immunologically active substance in a container contains an aqueous suspension of latex particles coated with the corresponding immunological reaction partner.
Im Falle eines indirekten Tests enthält die Reagenziengarnitur zur Bestimmung einer immunologisch aktiven Substanz in einem ersten Behälter eine Lösung des entsprechenden immunologischen Reaktionspartners (z.B. Antiserum) und in . einem zweiten Behälter eine wässrige Suspension von mit dem immunologisch aktiven Material beschichteten Latexteilchen.In the case of an indirect test, the reagent set for determining an immunologically active substance contains a solution of the corresponding immunological reaction partner (e.g. antiserum) in a first container and in. in a second container an aqueous suspension of latex particles coated with the immunologically active material.
In beiden Fällen kann die wässrige Suspension des an Latex gebundenen immunologisch aktiven Materials oder des an Latex gebundenen immunologischen Reaktionspartners in irgendeiner Konzentration vorhanden sein. Jedoch ist eine Konzentration von 0,5 bis 5 Gew.% bevorzugt.In either case, the aqueous suspension of the latex-bound immunologically active material or latex-linked immunological reactant can be present in any concentration. However, a concentration of 0.5 to 5% by weight is preferred.
Die Erfindung wird anhand der folgenden Beispielen veranschaulicht.The invention is illustrated by the following examples.
In einem Autoklaven von 25 Liter stellt man einen Latex von Kernpolymerisat her, wobei man verwendet:
- 4800 g entionisiertes Wasser
- 50 g Kaliumpersulfat
- 50 g Natriumpyrophosphat
- 10 g Natriumlaurylsulfat
- 50 g Natriummethallylsulfonat
- 100 g Acrylsäure
- 100 g Itaconsäure
- 2135 g Styrol
- 2865 g Butadien
- 4800 g of deionized water
- 50 g of potassium persulfate
- 50 g sodium pyrophosphate
- 10 g sodium lauryl sulfate
- 50 g sodium methallyl sulfonate
- 100 g acrylic acid
- 100 g itaconic acid
- 2135 g styrene
- 2865 g butadiene
Die Polymerisation wird bei 75°C unter Stickstoffatmosphäre ausgeführt, wobei die Monomeren im Verlauf von 7 Stunden kontinuierlich eingeführt werden und die Reaktion 8 Stunden lang fortgesetzt wird.The polymerization is carried out at 75 ° C. under a nitrogen atmosphere, the monomers being introduced continuously over a period of 7 hours and the reaction being continued for 8 hours.
Nach dem Abkühlen erhält man einen Latex vom pH = 2,5, dessen Konzentration an Polymerisatpartikeln 51 Gew.-% beträgt.After cooling, a latex of pH 2.5 is obtained, the concentration of polymer particles is 51% by weight.
Durch Elektronenmikroskopie stellt man fest, dass die Partikel einen mittleren Durchmesser von 0,145 µm haben; 90% der Partikel haben einen Durchmesser zwischen 0,14 und 0,15 µm.Electron microscopy shows that the particles have an average diameter of 0.145 µm; 90% of the particles have a diameter between 0.14 and 0.15 µm.
Die Zusammensetzung des Polymerisates ist im wesentlichen gleich wie diejenige der verwendeten Monomeren. Die Partikel tragen auf ihrer Oberfläche Carboxyl- und Sulfonatfunktionen, die durch konduktometrische Titration bestimmt werden.The composition of the polymer is essentially the same as that of the monomers used. The particles have carboxyl and sulfonate functions on their surface, which are determined by conductometric titration.
406 g des erhaltenen Latex und 1541 g entionisiertes Wasser werden in einen Reaktor eingeführt. Das Gemisch wird unter Rühren auf 70°C erhitzt; diese Temperatur wird während der ganzen Dauer der Reaktion aufrechterhalten.406 g of the latex obtained and 1541 g of deionized water are introduced into a reactor. The mixture is heated to 70 ° C. with stirring; this temperature is maintained throughout the reaction.
Sobald das Gemisch. 70°C erreicht hat, wird es unter Stickstoffatmosphäre gehalten; man gibt in 3 Stunden mit konstanter Geschwindigkeit gleichzeitig 1,25 g Natriumdihexylsulfosuccinat in 150 g Wasser, 0,20 g a,a'-Azobis-isobutyramidiniumchlorid in 210 g Wasser, 18 g Styrol, die 0,45 g p,p'-Dithiobisanilin enthalten, zu.Once the mixture. Reached 70 ° C, it is kept under nitrogen atmosphere; 1.25 g of sodium dihexylsulfosuccinate in 150 g of water, 0.20 ga, a'-azobis-isobutyramidinium chloride in 210 g water, 18 g styrene, the 0.45 gp, p'-dithiobisaniline are simultaneously added in 3 hours at a constant rate included, too.
Dann wird die Polymerisation 5 Stunden lang fortgesetzt. Das Gemisch wird danach abgekühlt.Then the polymerization is continued for 5 hours. The mixture is then cooled.
pH: 3,1
- Konzentration an Polymerisationspartikeln: 9,3 Gew.-%
- Elektrolytbeständigkeit: 5
- Concentration of polymerization particles: 9.3% by weight
- Electrolyte resistance: 5
mittlerer Durchmesser der Partikel: 0,15 m, wobei 90% einen Durchmesser zwischen 0,145 und 0,155 pm haben.average diameter of the particles: 0.15 m, 90% having a diameter between 0.145 and 0.155 pm.
Die Partikel tragen auf ihrer Oberfläche Carboxyl- und Sulfonatfunktionen, die durch konduktometrische Titration bestätigt werden, und Gruppen der Formel:
1 ml von dem oben hergestellten 10%igen Latex wird 20 ml Wasser zugesetzt und die Mischung 1 1/2 Stunden bei 35'000 g zentrifugiert. Der Ueberstand wird abdekantiert, der Rückstand in 20 ml Wasser aufgenommen und nochmals 1 1/2 Stunden bei 35'000 g zentrifugiert. Diese Operation wird zweimal wiederholt und der so erhaltene Latex wird in den folgenden Beispielen als "gewaschener Latex" bezeichnet.1 ml of the 10% latex produced above is added to 20 ml of water and the mixture is centrifuged at 35,000 g for 1 1/2 hours. The supernatant is decanted off, the residue is taken up in 20 ml of water and centrifuged again at 35,000 g for 1 1/2 hours. This operation is repeated twice and the latex thus obtained is referred to as "washed latex" in the following examples.
5 mg humanes Immunoglobulin G (Cohn Fraktion II) wird durch Erhitzen auf 60°C während 3 Stunden in 1 ml 0,1 M Glycin-HCl Puffer pH 4 denaturiert..5 mg of human immunoglobulin G (Cohn fraction II) is denatured by heating at 60 ° C. for 3 hours in 1 ml of 0.1 M glycine-HCl buffer pH 4.
1 ml gewaschener Latex wird 5 ml 0,05 M HC1 zugesetzt und die Mischung auf 0°C abgekühlt. Zu dieser Mischung wird 0,1 ml einer 0,01 M NaN02 Lösung gegeben und es wird 15 Minuten bei 0°C gerührt. Der diazotierte Latex wird bei 5°C 1 1/2 Stunden bei 35'000 g zentrifugiert und der Ueberstand abdekantiert. Der Rückstand wird in 5 ml eisgekühltem 0,1 M Glycin-HCl Puffer pH 4,0 aufgenommen und im gleichen Puffer 1 ml 0,5%iges denaturiertes humanes Immunoglobulin G zugegeben, 1 Stunde im Eisbad gerührt und anschliessend über Nacht bei 10° stehen gelassen. Der Latex wird 1 1/2 Stunden bei 35'000 g abzentrifugiert, der Ueberstand abdekantiert und der Rückstand zweimal mit je 25 ml 0,1 M Glycin-NaOH Puffer pH 8,2 gewaschen, durch Zentrifugieren und Aufschlämmen des Rückstandes. Dem Latex wird nun soviel Puffer zugesetzt, dass eine Lösung mit 30 mg/ml resultiert.1 ml of washed latex is added to 5 ml of 0.05 M HC1 and the mixture is cooled to 0 ° C. 0.1 ml of a 0.01 M NaNO 2 solution is added to this mixture and the mixture is stirred at 0 ° C. for 15 minutes. The diazotized latex is centrifuged at 5 ° C. for 1 1/2 hours at 35,000 g and the supernatant is decanted off. The residue is taken up in 5 ml of ice-cooled 0.1 M glycine-HCl buffer pH 4.0 and 1 ml of 0.5% denatured human immunoglobulin G is added in the same buffer, stirred for 1 hour in an ice bath and then standing overnight at 10 ° calmly. The latex is centrifuged off at 35,000 g for 1 1/2 hours, the supernatant is decanted off and the residue is washed twice with 25 ml of 0.1 M glycine-NaOH buffer pH 8.2, by centrifuging and slurrying the residue. So much buffer is now added to the latex that a solution with 30 mg / ml results.
Für den Röhrchen-Agglutinationstest wird der folgende Puffer verwendet: 7,5 g Glycin, 6,0 g CaCl2, 3 g Rinderalbumin, 1 g NaN3 gelöst in 1 Liter Wasser. Der pH-Wert wird mit NaOH auf 8,2 eingestellt. Für den Nachweis der Rheumafaktoren im Serum wird in einem kleinen Reagenzglas 20 µl Latex mit 3 ml Puffer verdünnt und 25 µl des zu untersuchenden Serums zugegeben. Nach dem Durchmischen werden die Röhrchen während 2 Stunden in einem Wärmeblock bei 37°C gehalten. Ein positives Serum agglutiniert unter diesen Bedingungen während ein negatives Kontrollsystem keine Agglutination zeigt.The following buffer is used for the tube agglutination test: 7.5 g glycine, 6.0 g CaCl 2 , 3 g bovine albumin, 1 g NaN 3 dissolved in 1 liter water. The pH is adjusted to 8.2 with NaOH. To detect the rheumatoid factors in the serum, 20 µl of latex is diluted with 3 ml of buffer in a small test tube and 25 µl of the serum to be examined is added. After mixing, the tubes are kept in a heat block at 37 ° C for 2 hours. A positive serum agglutinates under these conditions, while a negative control system shows no agglutination.
1 ml gewaschener Latex wird wie in Beispiel 1 hergestellt und diazotiert. Dem diazotierten Latexrückstand wird 5 ml eiskalter 0,1 M Glycin-NaOH Puffer pH 6,0 zugesetzt und 5 mg Ziegen anti-Humanalbumin Immunoglobulin G in 1 ml obigem Puffer gelöst zugegeben und die Mischung wird nach Rühren während 1 Stunde bei 10°C über Nacht stehen gelassen. Der Latex wird bei 35'000 g während 1 1/2 Stunden abzentrifugiert, der Ueberstand verworfen und das Sediment zweimal mit je 25 ml 0,1 M Glycin-NaOH pH 8,2 gewaschen. Nach dem Waschen wird der Latex mit soviel Puffer vermischt, dass eine 3%ige Lösung erhalten wird.1 ml of washed latex is prepared and diazotized as in Example 1. The diazotized latex residue becomes 5 ml ice-cold 0.1 M glycine-NaOH buffer pH 6.0 was added and 5 mg goat anti-human albumin immunoglobulin G dissolved in 1 ml of the above buffer were added and the mixture was left to stir overnight at 10 ° C. for 1 hour. The latex is centrifuged off at 35,000 g for 1 1/2 hours, the supernatant is discarded and the sediment is washed twice with 25 ml of 0.1 M glycine-NaOH pH 8.2. After washing, the latex is mixed with so much buffer that a 3% solution is obtained.
Für den Röhrchenagglutinationstest wird der folgende Puffer verwendet: 7,5 g Glycin, 6,0 g CaCl2, 3 g Rinderalbumin und 1,0 g NaN3 werden in 1 Liter Wasser gelöst und das pH mit Salzsäure auf 6,0 eingestellt. Es wird in kleinen Reagenzgläsern eine Konzentrationsreihe von Humanalbumin in 3 ml Puffer erstellt, je 20 µl Latex zugegeben und nach Durchmischen 2 Stunden bei 37°C in einem Wärmeblock gehalten.
Aus dieser Tabelle ist ersichtlich, dass mit dem so hergestellten Latex 0,05 µg Humanalbumin/ml bestimmt werden kann. ,From this table it can be seen that 0.05 µg human albumin / ml can be determined with the latex thus produced. ,
1 ml gewaschenem Latex von Beispiel 1 wird 5 ml 0,1 M Phosphatpuffer pH 5,0 zugesetzt und 5 mg Schaf anti-Human IgG Immunoglobulin G in 1 ml Puffer zugegeben und es wird gut gerührt Anschliessend wird 0,1 ml einer 0,01 M p-Phenyldiisothiocyanatlösung in Dimethylformamid zugegeben, 1 Stunde gerührt und bei Raumtemperatur über Nacht stehen gelassen. Der Latex wird 1 1/2 Stunden bei 35'000 g zentrifugiert, der Ueberstand verworfen und der Rückstand zweimal mit je 25 ml 0,1 M Glycin-NaOH Puffer pH 8,2 gewaschen. Für den Agglutinationstest wird der Latex in einer Konzentration von 30 mg/ml eingesetzt.1 ml of washed latex from Example 1 is added to 5 ml of 0.1 M phosphate buffer pH 5.0 and 5 mg of sheep anti-human IgG immunoglobulin G in 1 ml of buffer are added and the mixture is stirred well. Then 0.1 ml of a 0.01 M p-phenyldiisothiocyanate solution in dimethylformamide added, stirred for 1 hour and at Allow room temperature to stand overnight. The latex is centrifuged at 35,000 g for 1 1/2 hours, the supernatant is discarded and the residue is washed twice with 25 ml of 0.1 M glycine-NaOH buffer pH 8.2. The latex is used in a concentration of 30 mg / ml for the agglutination test.
Für den Röhrchenagglutinationstest wird ein 0,1 M Phosphatpuffer pH 6,0 mit 0,1% Rinderalbumin verwendet. Es wird eine Konzentrationsreihe von Human IgG in 3 ml Puffer hergestellt. Zu jedem Röhrchen werden 20 µl Latexreagens gegeben, durchmischt und 2 Stunden bei 37°C inkubiert im Wärmeblock.
Die Tabelle zeigt, dass mit diesem Latexreagens 0,1 µg/ml Human IgG noch bestimmt werden können.The table shows that 0.1 µg / ml human IgG can still be determined with this latex reagent.
36,8 mg Benzidin werden in 0,5 ml 2 NaCl gelöst und mit 7,5 ml Wasser verdünnt. Die Mischung wird im Eisbad gekühlt und unter Rühren 27,2 mg NaN02 in 2 ml Wasser zugetropft. Das so erhaltene bisdiazotierte Benzidin ist bei -20]-während Wochen haltbar.36.8 mg of benzidine are dissolved in 0.5 ml of 2 NaCl and diluted with 7.5 ml of water. The mixture is cooled in an ice bath and 27.2 mg of NaNO 2 in 2 ml of water are added dropwise with stirring. The bisdiazotized benzidine thus obtained can be kept at -20] for weeks.
1 ml gewaschener Latex von Beispiel 1 wird in 5 ml 0,1 M Phosphatpuffer pH 7,0 aufgenommen und 5 mg humanes Immunoglobulin G in 1 ml obigem Puffer zugegeben. Die Suspension wird auf 0° abgekühlt und unter Rühren 0,01 ml einer 0,02 M . bisdiazotierten Benzidinlösung zugegeben und anschliessend bei 10 0 über Nacht stehen gelassen. Der Latex wird bei 30'000 g während 1 1/2 Stunden zentrifugiert, der Ueberstand verworfen und das Sediment zweimal mit je 25 ml 0,1 M Glycin-NaOH pH 8,2 gewaschen. Nach dem Waschen wird der Latex wird soviel Puffer vermischt, dass eine 3%ige Lösung erhalten wird.1 ml of washed latex from Example 1 is taken up in 5 ml of 0.1 M phosphate buffer pH 7.0 and 5 mg of human immunoglobulin G in 1 ml of the above buffer is added. The suspension is cooled to 0 ° and 0.01 ml of a 0.02 M with stirring. bisdiazotierten benzidine solution was added and then at 1 0 0 left to stand overnight. The latex is centrifuged at 30,000 g for 1 1/2 hours, the supernatant is discarded and the sediment is washed twice with 25 ml of 0.1 M glycine-NaOH pH 8.2. After washing, the latex is mixed so much buffer that a 3% solution is obtained.
Für die Bestimmung von IgG im Inhibitionstest wird ein 0,1 M Phosphatpuffer pH 6,0 verwendet. In kleine Reagenzgläser wird je 3 ml eines 1/500 verdünnten Schaf anti-human IgG Serums und steigende Mengen von human IgG gegeben. Nach Inkubation während 15 Minuten bei 37°C wird in jedes Röhrchen 20 µl Latexreagenz gegeben und 3 Stunden bei 37°C inkubiert.
Es lassen sich mit dem oben beschriebenen Latexreagenz 0,16 µg human IgG/ml bestimmen.With the latex reagent described above, 0.16 µg human IgG / ml can be determined.
Claims (26)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH954277A CH628738A5 (en) | 1977-08-03 | 1977-08-03 | IMMUNOLOGICAL DIAGNOSTIC REAGENT. |
| CH9542/77 | 1977-08-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0000772A1 true EP0000772A1 (en) | 1979-02-21 |
Family
ID=4353079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP78100581A Withdrawn EP0000772A1 (en) | 1977-08-03 | 1978-08-02 | Immunological reagent consisting of special latex particles based on a vinylpolymer covered with an immunologically active material; process for its preparation; use of this reagent; analysing process using this reagent and test-kit containing this reagent |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US4226747A (en) |
| EP (1) | EP0000772A1 (en) |
| JP (1) | JPS5428816A (en) |
| AU (1) | AU528150B2 (en) |
| CA (1) | CA1103153A (en) |
| CH (1) | CH628738A5 (en) |
| DE (1) | DE2833886A1 (en) |
| DK (1) | DK343678A (en) |
| FR (1) | FR2399665A1 (en) |
| GB (1) | GB2001996B (en) |
| NL (1) | NL7808143A (en) |
| SE (1) | SE7808349L (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0008682A1 (en) * | 1978-08-02 | 1980-03-19 | F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft | Latex coated with protein material, process for the preparation of this latex, immunological reagent containing this latex, process for the preparation of this reagent, application of this reagent, testing procedure utilising this reagent and reagent kit containing this reagent |
| EP0019741A1 (en) * | 1979-05-07 | 1980-12-10 | BEHRINGWERKE Aktiengesellschaft | Latex reagent, process for its preparation, its application and an agent containing this reagent |
| FR2595826A1 (en) * | 1986-03-13 | 1987-09-18 | Lurhuma Zirimwabagabo | IMMUNOASSAY PRODUCT, PREPARATION METHOD THEREOF, USE THEREOF, IMMUNOGENIC COMPLEX COMPRISING THE SAME, AND USE OF THE COMPLEX |
Families Citing this family (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH628738A5 (en) * | 1977-08-03 | 1982-03-15 | Hoffmann La Roche | IMMUNOLOGICAL DIAGNOSTIC REAGENT. |
| DE3048883A1 (en) * | 1980-12-23 | 1982-07-15 | Boehringer Mannheim Gmbh, 6800 Mannheim | Hydrophilic latex particles prodn. by emulsion polymerisation - without added surfactant, useful as carriers for biological or immunological cpds. |
| JPS57168163A (en) * | 1981-04-10 | 1982-10-16 | Japan Synthetic Rubber Co Ltd | Carier for immunoserological inspection reagent |
| DE3116995A1 (en) * | 1981-04-29 | 1982-11-25 | Röhm GmbH, 6100 Darmstadt | LATEX FOR IMMOBILIZING BIOLOGICALLY EFFECTIVE SUBSTANCES |
| JPS585658A (en) * | 1981-07-02 | 1983-01-13 | Japan Synthetic Rubber Co Ltd | Carrier for immunity serological inspecting reagent |
| US4792527A (en) * | 1981-07-17 | 1988-12-20 | Toray Industries, Inc. | Method of assaying biologically active substances and labelling agents therefor |
| DE3130924A1 (en) * | 1981-08-05 | 1983-02-17 | Röhm GmbH, 6100 Darmstadt | SURFACE-BASED SYSTEMS FOR FIXING SUBSTRATES CONTAINING NUCLEOPHILE GROUPS |
| US4401765A (en) * | 1981-09-01 | 1983-08-30 | E. I. Du Pont De Nemours And Company | Covalently bonded high refractive index particle reagents and their use in light scattering immunoassays |
| JPS5847258A (en) * | 1981-09-01 | 1983-03-18 | イ−・アイ・デユポン・ド・ネモア−ス・アンド・コンパニ− | Granular reagent for light-scattering immunoassay |
| US4480042A (en) * | 1981-10-28 | 1984-10-30 | E. I. Du Pont De Nemours And Company | Covalently bonded high refractive index particle reagents and their use in light scattering immunoassays |
| JPS5897656A (en) * | 1981-12-07 | 1983-06-10 | Sekisui Chem Co Ltd | Latex for diagnosis reagent |
| US4713350A (en) * | 1983-10-24 | 1987-12-15 | Technicon Instruments Corporation | Hydrophilic assay reagent containing one member of specific binding pair |
| US4654325A (en) * | 1984-05-24 | 1987-03-31 | Selenke William M | Medicament for reducing nephrotoxicity caused by positively charged agents such as aminoglycosides and methods of use thereof |
| JPH0692970B2 (en) * | 1986-02-28 | 1994-11-16 | 日本合成ゴム株式会社 | Method for producing carrier particles for diagnostic agent |
| US5166077A (en) * | 1987-04-30 | 1992-11-24 | Nitto Denko Corporation | Latex for immobilization of physiologically active substances for immuno nephelometry |
| DE69126142T2 (en) * | 1990-09-26 | 1997-09-11 | Akers Lab Inc | IMPROVED LIGAND DETERMINATION PROCEDURE |
| US5231035A (en) * | 1991-01-11 | 1993-07-27 | Akers Research Corporation | Latex agglutination assay |
| US5973029A (en) * | 1993-10-12 | 1999-10-26 | The Sherwin-Williams Company | Corrosion-resistant waterborne paints |
| US5432210A (en) * | 1993-11-22 | 1995-07-11 | Rohm And Haas Company | Polymer particles and method for preparing by polymerization of encapsulated monomers |
| US5981296A (en) * | 1997-02-18 | 1999-11-09 | Dade Behring Inc. | Stabilization of particle reagents |
| IT1291164B1 (en) † | 1997-03-04 | 1998-12-29 | Coral Spa | UNIVERSAL DUCT FOR THE CONVEYANCE OF HARMFUL SMOKES OR GAS FROM A WORKING PLACE. |
| ATE458762T1 (en) * | 2005-07-01 | 2010-03-15 | Hoffmann La Roche | CARBOXYLATED LATEX PARTICLES |
| WO2008024509A2 (en) | 2006-08-24 | 2008-02-28 | Mallard Creek Polymers, Inc. | Cationic latex as a carrier for bioactive ingredients and methods for making and using the same |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1806418A1 (en) * | 1967-11-01 | 1969-06-26 | Miles Lab | Protein-like reagent and manufacturing process therefor |
| DE2204684A1 (en) * | 1971-02-01 | 1972-08-17 | Hoffmann La Roche | Method for the detection of antibodies or antigens in a body fluid |
| US3857931A (en) * | 1971-02-01 | 1974-12-31 | Hoffmann La Roche | Latex polymer reagents for diagnostic tests |
| FR2331567A1 (en) * | 1975-11-13 | 1977-06-10 | Inst Nat Sante Rech Med | Fixation of organic cpds. contg. sugar residues - by oxidn. and coupling to a support contg. amino gps. |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4118349A (en) * | 1971-05-12 | 1978-10-03 | Behringwerke Aktiengesellschaft | Process for the manufacture of polystyrene latex compounds |
| US3896217A (en) * | 1973-03-19 | 1975-07-22 | Summa Corp | Method and apparatus for radioimmunoassay with regeneration of immunoadsorbent |
| FR2321519A1 (en) * | 1975-08-22 | 1977-03-18 | Rhone Poulenc Ind | STYRENIC POLYMER LATEX |
| US4140662A (en) * | 1977-03-25 | 1979-02-20 | Ortho Diagnostics, Inc. | Attachment of proteins to inert particles |
| US4134872A (en) * | 1977-05-20 | 1979-01-16 | The Dow Chemical Company | Heterogeneous polymer particles comprising an interpolymer domain of a monovinylidene aromatic monomer, an open chain aliphatic conjugated diene and a monoethylenically unsaturated acid |
| CH628738A5 (en) * | 1977-08-03 | 1982-03-15 | Hoffmann La Roche | IMMUNOLOGICAL DIAGNOSTIC REAGENT. |
| US4156669A (en) * | 1978-04-24 | 1979-05-29 | The Dow Chemical Company | Latexes of encapsulated vinylidene chloride copolymer particles |
-
1977
- 1977-08-03 CH CH954277A patent/CH628738A5/en not_active IP Right Cessation
-
1978
- 1978-07-12 CA CA307,224A patent/CA1103153A/en not_active Expired
- 1978-07-27 AU AU38407/78A patent/AU528150B2/en not_active Expired
- 1978-07-31 US US05/929,410 patent/US4226747A/en not_active Expired - Lifetime
- 1978-08-01 FR FR7822676A patent/FR2399665A1/en active Granted
- 1978-08-02 SE SE7808349A patent/SE7808349L/en unknown
- 1978-08-02 DE DE19782833886 patent/DE2833886A1/en not_active Withdrawn
- 1978-08-02 EP EP78100581A patent/EP0000772A1/en not_active Withdrawn
- 1978-08-02 DK DK343678A patent/DK343678A/en not_active Application Discontinuation
- 1978-08-02 GB GB7831927A patent/GB2001996B/en not_active Expired
- 1978-08-02 JP JP9373978A patent/JPS5428816A/en active Pending
- 1978-08-02 NL NL787808143A patent/NL7808143A/en not_active Application Discontinuation
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1806418A1 (en) * | 1967-11-01 | 1969-06-26 | Miles Lab | Protein-like reagent and manufacturing process therefor |
| DE2204684A1 (en) * | 1971-02-01 | 1972-08-17 | Hoffmann La Roche | Method for the detection of antibodies or antigens in a body fluid |
| US3857931A (en) * | 1971-02-01 | 1974-12-31 | Hoffmann La Roche | Latex polymer reagents for diagnostic tests |
| FR2331567A1 (en) * | 1975-11-13 | 1977-06-10 | Inst Nat Sante Rech Med | Fixation of organic cpds. contg. sugar residues - by oxidn. and coupling to a support contg. amino gps. |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0008682A1 (en) * | 1978-08-02 | 1980-03-19 | F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft | Latex coated with protein material, process for the preparation of this latex, immunological reagent containing this latex, process for the preparation of this reagent, application of this reagent, testing procedure utilising this reagent and reagent kit containing this reagent |
| EP0019741A1 (en) * | 1979-05-07 | 1980-12-10 | BEHRINGWERKE Aktiengesellschaft | Latex reagent, process for its preparation, its application and an agent containing this reagent |
| FR2595826A1 (en) * | 1986-03-13 | 1987-09-18 | Lurhuma Zirimwabagabo | IMMUNOASSAY PRODUCT, PREPARATION METHOD THEREOF, USE THEREOF, IMMUNOGENIC COMPLEX COMPRISING THE SAME, AND USE OF THE COMPLEX |
| EP0238396A1 (en) * | 1986-03-13 | 1987-09-23 | Zirimwabagabo Lurhuma | Immunoassay product, process for its preparation, its use, an immunocomplex containing it and use of this complex |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2001996B (en) | 1982-03-24 |
| SE7808349L (en) | 1979-02-04 |
| JPS5428816A (en) | 1979-03-03 |
| CA1103153A (en) | 1981-06-16 |
| DE2833886A1 (en) | 1979-02-22 |
| FR2399665A1 (en) | 1979-03-02 |
| AU3840778A (en) | 1980-01-31 |
| DK343678A (en) | 1979-02-04 |
| NL7808143A (en) | 1979-02-06 |
| US4226747A (en) | 1980-10-07 |
| GB2001996A (en) | 1979-02-14 |
| CH628738A5 (en) | 1982-03-15 |
| FR2399665B1 (en) | 1982-11-05 |
| AU528150B2 (en) | 1983-04-14 |
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Legal Events
| Date | Code | Title | Description |
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| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| AK | Designated contracting states |
Designated state(s): BE CH DE FR GB NL SE |
|
| 17P | Request for examination filed | ||
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
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| 18D | Application deemed to be withdrawn |
Effective date: 19801203 |
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| RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: GAETANO, RONCARI, DR. |