WO2010055883A1 - 抗リン脂質抗体測定試薬に用いる不溶性担体、抗リン脂質抗体測定試薬、及び、抗リン脂質抗体の測定方法 - Google Patents
抗リン脂質抗体測定試薬に用いる不溶性担体、抗リン脂質抗体測定試薬、及び、抗リン脂質抗体の測定方法 Download PDFInfo
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- WO2010055883A1 WO2010055883A1 PCT/JP2009/069273 JP2009069273W WO2010055883A1 WO 2010055883 A1 WO2010055883 A1 WO 2010055883A1 JP 2009069273 W JP2009069273 W JP 2009069273W WO 2010055883 A1 WO2010055883 A1 WO 2010055883A1
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- WIPO (PCT)
- Prior art keywords
- insoluble carrier
- reagent
- measuring
- antiphospholipid antibody
- measurement reagent
- Prior art date
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 68
- 238000005259 measurement Methods 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000001488 sodium phosphate Substances 0.000 claims abstract description 10
- 229910000162 sodium phosphate Inorganic materials 0.000 claims abstract description 10
- 239000007787 solid Substances 0.000 claims abstract description 10
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims abstract description 10
- 239000002245 particle Substances 0.000 claims description 80
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 10
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 8
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
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- MNCGMVDMOKPCSQ-UHFFFAOYSA-M sodium;2-phenylethenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C=CC1=CC=CC=C1 MNCGMVDMOKPCSQ-UHFFFAOYSA-M 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
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- 235000012000 cholesterol Nutrition 0.000 description 5
- 238000013329 compounding Methods 0.000 description 5
- ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2,3-di(octadeca-9,12-dienoyloxy)propoxy-oxidophosphoryl]oxy-2-hydroxypropyl] 2,3-di(octadeca-9,12-dienoyloxy)propyl phosphate Chemical compound [Na+].[Na+].CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COP([O-])(=O)OCC(O)COP([O-])(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 description 5
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- 238000000691 measurement method Methods 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
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- 150000003839 salts Chemical class 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
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- AGBXYHCHUYARJY-UHFFFAOYSA-N 2-phenylethenesulfonic acid Chemical compound OS(=O)(=O)C=CC1=CC=CC=C1 AGBXYHCHUYARJY-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
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- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
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- 229920001218 Pullulan Polymers 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- JRKICGRDRMAZLK-UHFFFAOYSA-L persulfate group Chemical class S(=O)(=O)([O-])OOS(=O)(=O)[O-] JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 2
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- KTZVZZJJVJQZHV-UHFFFAOYSA-N 1-chloro-4-ethenylbenzene Chemical compound ClC1=CC=C(C=C)C=C1 KTZVZZJJVJQZHV-UHFFFAOYSA-N 0.000 description 1
- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 description 1
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 1
- IWTYTFSSTWXZFU-UHFFFAOYSA-N 3-chloroprop-1-enylbenzene Chemical compound ClCC=CC1=CC=CC=C1 IWTYTFSSTWXZFU-UHFFFAOYSA-N 0.000 description 1
- JLBJTVDPSNHSKJ-UHFFFAOYSA-N 4-Methylstyrene Chemical compound CC1=CC=C(C=C)C=C1 JLBJTVDPSNHSKJ-UHFFFAOYSA-N 0.000 description 1
- 229920002126 Acrylic acid copolymer Polymers 0.000 description 1
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- 241000894006 Bacteria Species 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 229920000298 Cellophane Polymers 0.000 description 1
- 235000019743 Choline chloride Nutrition 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- 235000010469 Glycine max Nutrition 0.000 description 1
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- XECAHXYUAAWDEL-UHFFFAOYSA-N acrylonitrile butadiene styrene Chemical compound C=CC=C.C=CC#N.C=CC1=CC=CC=C1 XECAHXYUAAWDEL-UHFFFAOYSA-N 0.000 description 1
- 229920000122 acrylonitrile butadiene styrene Polymers 0.000 description 1
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- XYLMUPLGERFSHI-UHFFFAOYSA-N alpha-Methylstyrene Chemical compound CC(=C)C1=CC=CC=C1 XYLMUPLGERFSHI-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
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- MPMBRWOOISTHJV-UHFFFAOYSA-N but-1-enylbenzene Chemical compound CCC=CC1=CC=CC=C1 MPMBRWOOISTHJV-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- LEDCJBCTEYLBJO-UHFFFAOYSA-N ethyl 2-phenylethenesulfonate Chemical compound CCOS(=O)(=O)C=CC1=CC=CC=C1 LEDCJBCTEYLBJO-UHFFFAOYSA-N 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 150000002237 fumaric acid derivatives Chemical class 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910003002 lithium salt Inorganic materials 0.000 description 1
- 159000000002 lithium salts Chemical class 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 150000002688 maleic acid derivatives Chemical class 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
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- 238000010992 reflux Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
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- 239000006228 supernatant Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229920001567 vinyl ester resin Polymers 0.000 description 1
- -1 vinyl halide Chemical class 0.000 description 1
Images
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
-
- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
-
- 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
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Definitions
- the present invention relates to an insoluble carrier used for highly reactive antiphospholipid antibody measurement reagents.
- the present invention also relates to an antiphospholipid antibody measurement reagent and a method of measuring an antiphospholipid antibody.
- An immunological measurement method is employed as a method of measuring trace substances contained in blood, urine and the like.
- the immunological measurement method is based on the specific strong binding of the antigen-antibody reaction, and it is possible to measure the target substance specifically and sensitively even from a sample in which various substances are mixed.
- the zeta potential of the insoluble carrier used in the immunological measurement method is measured under certain conditions, and the carrier in the range of -20 mV or more and less than 0 mV is used.
- the amount of sulfonic acid groups on the surface of latex particles used in the method of selecting a large amount of physically or physically adsorbing an antigen or antibody (Patent Document 1) or the method of immunological measurement is 0.005 to 0.7 ⁇ mol / m 2
- carrier with which the range was controlled are proposed.
- An object of the present invention is to provide an insoluble carrier used for a highly reactive antiphospholipid antibody measurement reagent. Another object of the present invention is to provide an antiphospholipid antibody measurement reagent and a method of measuring an antiphospholipid antibody.
- the inventors of the present invention have a method of adsorbing a phospholipid to an insoluble carrier to an insoluble carrier having a zeta potential in the range of ⁇ 20 mV or more and less than 0 mV, unlike the case where an antigen or antibody consisting of ordinary protein is adsorbed to an insoluble carrier. It has been found that there is a problem that antiphospholipid antibodies can not be measured with sufficient sensitivity. As a result of intensive studies, the present inventors compared the insoluble carriers having the same particle size but different zeta potentials when using phospholipids with few hydrophilic moieties adsorbed to the insoluble carrier, it is better to use an insoluble carrier having a low zeta potential.
- the present invention relates to an insoluble carrier for use in an antiphospholipid antibody measurement reagent, wherein the solid concentration is adjusted to 0.1% in a 20 mmol / L aqueous sodium phosphate solution (pH 7.4). It is an insoluble carrier used for the antiphospholipid antibody measurement reagent whose zeta potential in the case of turbidity is less than -45 mV.
- the present invention is also a reagent for measuring an antiphospholipid antibody using the insoluble carrier, and a method for measuring an antiphospholipid antibody using the insoluble carrier.
- the highly reactive antiphospholipid antibody measurement reagent can be obtained by using an insoluble carrier having a low zeta potential.
- the zeta potential approaches 0, the repulsion of the insoluble carrier decreases and aggregation tends to occur, so it is said that the insoluble carrier with a zeta potential near 0 has higher reactivity.
- the distance between the antigen (or antibody) to be adsorbed to the insoluble carrier and the antibody (or antigen) that reacts with it is too short, it is difficult for the antigen-antibody reaction to occur. Is considered to occur.
- the insoluble carrier used for the reagent for measuring an antiphospholipid antibody of the present invention has a zeta potential when suspended in a 20 mmol / L aqueous sodium phosphate solution (pH 7.4) so that the solid concentration becomes 0.1%. Insoluble carrier less than 45mV. When the zeta potential is less than ⁇ 45 mV, the antiphospholipid antibody measurement reagent using the insoluble carrier used for the antiphospholipid antibody measurement reagent of the present invention has high reactivity.
- the lower limit of the zeta potential is not particularly limited, but substantially the lower limit is about -100 mV.
- the zeta potential is preferably ⁇ 74 mV or less.
- the insoluble carrier used for the antiphospholipid antibody measurement reagent of the present invention is not particularly limited, and examples thereof include organic polymer powder, microorganisms, blood cells, cell membrane fragments and the like. Among them, organic polymer powder is preferable. Examples of the organic polymer powder include natural polymer powder and synthetic polymer powder.
- the natural polymer powder is not particularly limited, and examples thereof include insoluble agarose, cellulose, insoluble dextran and the like.
- the synthetic polymer powder is not particularly limited.
- polystyrene, styrene-sulfonic acid (salt) copolymer, styrene- (meth) acrylic acid copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride- Examples include meta) acrylic acid ester copolymer, vinyl acetate- (meth) acrylic acid ester copolymer and the like.
- the insoluble carrier used for the antiphospholipid antibody measurement reagent of the present invention may be an insoluble carrier having a sulfonic acid group or a carboxyl group introduced on the surface.
- the synthetic polymer fine particles be latex particles uniformly dispersed in a water medium.
- the latex particles are composed of a copolymer of a polymerizable monomer having a phenyl group and a polymerizable monomer having a phenyl group and a sulfonic acid group.
- the polymerizable monomer having a phenyl group is not particularly limited, and examples thereof include styrene, divinylbenzene, ethylstyrene, ⁇ -methylstyrene, p-methylstyrene, p-chlorostyrene, chloromethylstyrene and the like.
- These polymerizable monomers having a phenyl group may be used alone or in combination of two or more. Among them, styrene is preferred.
- the polymerizable monomer having a phenyl group and a sulfonic acid group is not particularly limited as long as it is a monomer capable of containing a sulfonic acid group on the surface of the carrier particle after polymerization, for example, a styrene sulfonate, Examples thereof include divinyl benzene sulfonate, ethyl styrene sulfonate, ⁇ -methyl sulfonate and the like.
- the salt in this case is not specifically limited, For example, sodium salt, potassium salt, lithium salt, ammonium salt etc. are mentioned.
- polymerizable monomers having a phenyl group and a sulfonic acid group may be used alone or in combination of two or more.
- styrene sulfonate is preferable, and sodium styrene sulfonate is more preferable.
- the latex particles are obtained by copolymerizing the polymerizable monomer having the phenyl group with the polymerizable monomer having the phenyl group and the sulfonic acid group.
- a conventionally known method can be used as a method of copolymerizing the polymerizable monomer having a phenyl group and the polymerizable monomer having a phenyl group and a sulfonic acid group.
- water is used as a solvent.
- the polymerizable monomer having a phenyl group Under the nitrogen atmosphere, the polymerizable monomer having a phenyl group, the polymerizable monomer having a phenyl group and a sulfonic acid group, a polymerization initiator, and an emulsifying agent as needed are added in a reaction vessel. And the like.
- the polymerization temperature at the time of copolymerizing the polymerizable monomer which has the said phenyl group, and the polymerizable monomer which has the said phenyl group and a sulfonic acid group is not specifically limited, A preferable minimum is 50 degreeC and a preferable upper limit is 100. ° C. If the polymerization temperature is less than 50 ° C., the polymerization reaction may not proceed sufficiently. When the polymerization temperature exceeds 100 ° C., the polymerization rate may be too fast, and it may be difficult to control the particle size. A more preferable lower limit of the polymerization temperature is 60 ° C., and a more preferable upper limit is 85 ° C.
- the polymerization time is usually 5 to 50 hours depending on the composition and concentration of the polymerizable monomer and the conditions such as the polymerization initiator.
- the blending amount of the polymerizable monomer having a phenyl group and a sulfonic acid group with respect to the polymerizable monomer having a phenyl group is determined in consideration of the sulfonic acid group amount on the particle surface obtained by copolymerization and the particle diameter. Need to be set.
- the insoluble carrier used in the reagent for measuring an antiphospholipid antibody of the present invention does not adsorb a conventional protein-derived antigen or antibody, but adsorbs a phospholipid with a low charge.
- the amount of sulfonic acid groups on the surface of the latex particles is preferably 0.1 ⁇ mol / m 2 or more. Therefore, the blending amount of the polymerizable monomer having a phenyl group and a sulfonic acid group with respect to the polymerizable monomer having a phenyl group is 0.1 to 0.7 ⁇ mol of a sulfonic acid group on the particle surface after copolymerization.
- the amount of sulfonic acid groups on the surface of the latex particle can be determined by the conductivity titration method (Journal of Colloid and Interface Sciences. 49 (3) 425, 1974), and this value can be determined from the particle diameter obtained.
- the amount of sulfonic acid group per unit area can be calculated by dividing by the total surface area of particles calculated.
- the said polymerization initiator is not specifically limited, For example, a persulfate salt etc. are mentioned. Although the above-mentioned persulfates are not particularly limited, potassium persulfate, sodium persulfate, ammonium persulfate and the like are preferable.
- the blending amount of the polymerization initiator is not particularly limited, but it is usually in the range of 0.01 to 1% by weight with respect to the amount of the polymerizable monomer.
- the above-mentioned emulsifier is not usually used because it has the disadvantage of inhibiting measurement accuracy if the above-mentioned latex particles contain the emulsifier, it is necessary for adjusting the amount of sulfonic acid groups on the surface of the above-mentioned latex particles. If necessary, it can be used.
- the compounding amount of the above-mentioned emulsifier is not particularly limited, but in consideration of removal by a post-treatment step after polymerization, the upper limit is preferably 1% by weight, more preferably the upper limit to the polymerizable monomer having a phenyl group. The upper limit is preferably 0.5% by weight, more preferably 0.02% by weight. The preferable lower limit of the blending amount of the emulsifier is 0.01% by weight.
- a polymerizable unsaturated monomer may be further added.
- the polymerizable unsaturated monomer is not particularly limited as long as it can be used for ordinary radical polymerization, and, for example, (meth) acrylic acid, (meth) acrylic acid ester, styrene derivative, (meth) acrylonitrile, (meth) Acrylic acid amide, vinyl halide, vinyl ester, (meth) acrolein, maleic acid derivative, fumaric acid derivative and the like can be mentioned.
- (meth) acrylic acid means acrylic acid or methacrylic acid.
- salts may be added.
- the above salts are not particularly limited as long as they can be used for ordinary radical polymerization, for example, magnesium sulfate, calcium sulfate, disodium sulfate, dipotassium sulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, dihydrogen phosphate Potassium, dipotassium hydrogen phosphate, sodium chloride, potassium chloride and the like can be mentioned.
- the average particle size of the latex particles used for the measurement may be appropriately selected depending on the measuring method and the measuring device to be used, but the generally preferred lower limit is 0.01 ⁇ m and the preferred upper limit is 1.5 ⁇ m. If the average particle size of the latex particles is less than 0.01 ⁇ m, the amount of optical change due to aggregation is too small to obtain the sensitivity required for measurement, and many times during centrifugation at the time of reagent preparation Reagent cost may be high. When the average particle diameter of the latex particles exceeds 1.5 ⁇ m, the optical change due to the aggregation of the latex particles exceeds the measurable range when the concentration of the substance to be measured is high, and the amount of the substance to be measured is increased. Optical change may not be obtained.
- the lower limit of the average particle size of the latex particles used in the present invention is preferably 0.2 ⁇ m, and 0.5 ⁇ m, more preferably The lower limit is 0.3 ⁇ m, and the more preferable upper limit is 0.4 ⁇ m.
- the average particle size can be determined by image analysis using a transmission electron microscope.
- the coefficient of variation (hereinafter also referred to as CV value (%)) of the particle diameter of the latex particles is not particularly limited, but a preferable upper limit is 10%. If the CV value (%) of the particle diameter of the latex particles exceeds 10%, the lot reproducibility at the time of preparation of the reagent may be poor, and the reproducibility of the measurement reagent may be reduced. A more preferable upper limit of the CV value (%) of the particle diameter of the latex particle is 5%, and a still more preferable upper limit is 3%.
- the above latex particles have a zeta potential of less than -45 mV when suspended not only in one type of particles but in a 20 mmol / L aqueous sodium phosphate solution (pH 7.4) so that the solid concentration becomes 0.1%. If so, two or more types of latex particles different in zeta potential may be used.
- an antigen-antibody reaction can be measured with high sensitivity and high accuracy over a wide concentration range from low concentration to high concentration, and in particular, spectrophotometer, turbidity meter, light scattering
- a measurement reagent suitable for optical measurement devices such as measurement devices can be obtained.
- An anti-phospholipid antibody measurement reagent for use in anti-phospholipid antibody measurement comprising an insoluble carrier carrying a phospholipid antigen and a buffer solution, wherein the insoluble carrier before carrying the phospholipid antigen is 20 mmol / L
- An antiphospholipid antibody measurement reagent having a zeta potential of less than -45 mV when suspended in an aqueous solution of sodium phosphate (pH 7.4) to have a solid concentration of 0.1% is also one of the present invention. is there.
- the antiphospholipid antibody measurement reagent of the present invention contains an insoluble carrier carrying a phospholipid antigen.
- the phospholipid antigen is not particularly limited, but for example, a phospholipid antigen consisting of cardiolipin, phosphatidyl choline and cholesterol is preferable.
- the cardiolipin is preferably purified from bovine heart, but may be chemically synthesized.
- the phosphatidylcholine is preferably purified from chicken egg yolk, but lecithin having a phosphatidylcholine content of 60 to 80% may be used.
- those extracted from bovine heart, soybean, etc., or those chemically synthesized may be used.
- the cholesterol may be of animal origin or may be chemically synthesized.
- the mixing ratio of the above cardiolipin, phosphatidyl choline and cholesterol is not particularly limited, but it is preferable that 8 to 12 mg of phosphatidyl choline and 1 to 5 mg of cholesterol are contained with respect to 1 mg of cardiolipin.
- the method for supporting the above-mentioned phospholipid antigen on the above-mentioned insoluble carrier is not particularly limited, and examples thereof include a method for supporting the above-mentioned phospholipid antigen by physical and / or chemical bond by a conventionally known method.
- the antiphospholipid antibody measurement reagent of the present invention contains a buffer.
- the buffer solution has a role of dispersing or suspending the latex particle supporting the phospholipid antigen.
- the buffer is not particularly limited, and examples thereof include phosphate buffer, glycine buffer, tris salt buffer, Good buffer and the like.
- the pH of the buffer solution is not particularly limited, but the preferable lower limit is 5.5, the preferable upper limit is 8.5, and the more preferable lower limit is 6.5.
- the anti-phospholipid antibody measurement reagent of the present invention may contain a water-soluble polymer for the purpose of improving the measurement sensitivity and promoting the antigen-antibody reaction.
- the water-soluble polymer is not particularly limited, and examples thereof include pullulan and polyvinyl pyrrolidone.
- a method for measuring an antiphospholipid antibody using an antiphospholipid antibody measurement reagent comprising the steps of measuring a lipid antibody in a sample, the insoluble carrier prior to loading of the phospholipid antigen comprising
- a method of measuring an antiphospholipid antibody having a zeta potential of less than ⁇ 45 mV when suspended in a 20 mmol / L aqueous sodium phosphate solution (pH 7.4) to a solid content concentration of 0.1% It is one of the present invention.
- the method of optically measuring the degree of aggregation is not particularly limited, and a conventionally known method may be used, for example, the size of the insoluble carrier particles used, the selection of the concentration, the setting of the reaction time, the scattered light intensity, Methods of measuring increase and decrease of absorbance and transmitted light intensity can be mentioned. Moreover, it is also possible to use these methods together.
- the wavelength of light at the time of the above measurement is preferably 300 to 900 nm.
- the highest concentration of 8.0 R. U When using the method of measuring the increase or decrease of the absorbance, in order to make an accurate measurement, the highest concentration of 8.0 R. U.
- the reactivity is required to be at least 250 mAbs as the change in absorbance at (a unit of syphilis-positive antibody titer, diagnosed as syphilis-positive at 1.0 R.U. or higher).
- 8.0R. U In the case where the amount of change in absorbance of the antibody is 250 mAbs, the 1.0R. U. The amount of change in absorbance is about 30 mAbs. 1.0R. U. When the amount of change in absorbance is less than 30 mAbs, the reproducibility of the data is significantly reduced, and it can not be accurately determined whether it is negative or positive.
- the apparatus used for the method of optically measuring the degree of aggregation is not particularly limited, and optical instruments capable of detecting scattered light intensity, transmitted light intensity, absorbance and the like can be mentioned, and commonly used biochemical automatic analysis Any machine can be used.
- the method of visually observing the degree of aggregation is usually a method of mixing the sample and the antiphospholipid antibody measurement reagent of the present invention on a determination plate, shaking the mixed solution, and determining the presence or absence of aggregation, etc. Can be used.
- a method of imaging the aggregation state with a video camera or the like and performing image processing other than the method by visual observation.
- an insoluble carrier used for a highly reactive antiphospholipid antibody measurement reagent it is possible to provide an antiphospholipid antibody measurement reagent and a method of measuring an antiphospholipid antibody.
- Latex LotA (Preparation of latex particles)
- a glass reaction vessel (2 L in volume) equipped with a stirrer, a condenser for reflux, a temperature detector, a nitrogen inlet tube and a jacket, 1100 g of distilled water, 180 g of styrene, 0.04 g of sodium styrene sulfonate, and 26 g of distilled water
- An aqueous solution in which 0.8 g of potassium persulfate was dissolved was charged, the inside of the vessel was replaced with nitrogen gas, and polymerization was carried out for 48 hours while stirring at 70 ° C. After completion of the polymerization, the above solution was filtered with filter paper to take out latex particles.
- the particle diameter of the obtained latex particles, the amount of sulfonic acid groups on the surface and the zeta potential were measured by the following method.
- the latex particles were photographed at a magnification of 10000 using a transmission electron microscope ("JEM-1010" manufactured by JEOL Ltd.), and the particle diameter was measured by image analysis of at least 100 particles. The average particle size obtained was 0.4 ⁇ m.
- the latex particles were dialyzed against purified water with a cellophane tube dialysis membrane for 48 hours to remove residual monomers. The particles were collected in a four-necked glass container to a dry weight of 10 g, diluted to 150 mL with distilled water and stirred using a stirrer tip. This was solution A. Next, 0.01N sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) is set in the attached device ATB-310 electric burette of the potentiometric electric conductivity titration processor (manufactured by Kyoto Denshi Kogyo, "AT-310").
- the conductivity electrode was immersed in the solution A, and a nitrogen introducing tube, a degassing tube and a pH electrode were set. Then, 0.01 N sodium hydroxide is dropped (0.05 mL is adjusted according to the amount of sulfonic acid measured in the range of 150 seconds to 500 seconds), and the potentiometric electric conductivity titration apparatus (manufactured by Kyoto Denshi Kogyo Co., Ltd., “AT The equivalent point was measured from the amount of change in conductivity using “ ⁇ 310”) to calculate the target amount of sulfonic acid groups. The amount of sulfonic acid groups obtained was 0.28 ⁇ mol / m 2 .
- the latex particles were adjusted to a solid content concentration of 0.1% in a 20 mmol / L aqueous sodium phosphate solution (pH 7.4), and this was used as a zeta potential measurement sample.
- 750 ⁇ L of the measurement sample was dispensed into a capillary cell for measuring zeta potential using a zeta potential measurement device (“Zetasizer Nano ZEN 3600” manufactured by Malvern Instruments Ltd.), and the zeta potential was measured at a measurement temperature of 37 ° C. The obtained zeta potential was -74 mV.
- Latex LotB Latex particles were produced in the same manner as Lot A, except that the blending amount of sodium styrene sulfonate was 0.08 g. The evaluation was performed in the same manner as for LotA, and the average particle size of the obtained latex particles was 0.3 ⁇ m, the amount of sulfonic acid groups was 0.23 ⁇ mol / m 2 , and the zeta potential was ⁇ 77 mV.
- Latex LotC The compounding amount of distilled water is 1020 g, the compounding amount of sodium styrene sulfonate is 0.25 g, and 0.4 g of potassium persulfate is dissolved in 13 g of distilled water instead of an aqueous solution in which 0.8 g of potassium persulfate is dissolved in 26 g of distilled water Using the aqueous solution thus prepared, latex particles were produced in the same manner as Lot A except that 80 g of 0.1 mol / L dipotassium hydrogen phosphate was further added.
- the evaluation was performed in the same manner as for LotA, and the average particle size of the obtained latex particles was 0.3 ⁇ m, the amount of sulfonic acid groups was 0.16 ⁇ mol / m 2 , and the zeta potential was ⁇ 88 mV.
- Latex LotD The compounding amount of distilled water is 1020 g, the compounding amount of sodium styrene sulfonate is 0.20 g, and 0.6 g of potassium persulfate is dissolved in 16 g of distilled water instead of an aqueous solution in which 0.8 g of potassium persulfate is dissolved in 26 g of distilled water Using the aqueous solution thus prepared, latex particles were produced in the same manner as Lot A except that 80 g of 0.1 mol / L dipotassium hydrogen phosphate was further added.
- the evaluation was performed in the same manner as for LotA, and the average particle size of the obtained latex particles was 0.4 ⁇ m, the amount of sulfonic acid groups was 0.18 ⁇ mol / m 2 , and the zeta potential was ⁇ 86 mV.
- Latex LotE An aqueous solution of 0.1 g of potassium persulfate dissolved in 4 g of distilled water was used instead of an aqueous solution in which 0.8 g of potassium persulfate was dissolved in 26 g of distilled water, with 0.01 g of sodium styrene sulfonate blended. Latex particles were prepared in the same manner as LotA. The average particle diameter of the obtained latex particles was 0.4 ⁇ m, the amount of sulfonic acid groups was 0.06 ⁇ mol / m 2 , and the zeta potential was ⁇ 29 mV.
- Example 1 (1) Preparation of buffer solution (first reagent) 25 mmol /% containing 1.2 (W / V)% pullulan (manufactured by Hayashibara), 1.0 (W / V)% bovine serum albumin (BSA) To 100 mL of L phosphate buffer (pH 6.5), 0.9 g of sodium chloride and 0.1 g of sodium azide were added to make a buffer solution (first reagent).
- first reagent 25 mmol /% containing 1.2 (W / V)% pullulan (manufactured by Hayashibara), 1.0 (W / V)% bovine serum albumin (BSA)
- L phosphate buffer pH 6.5
- 0.9 g of sodium chloride and 0.1 g of sodium azide were added to make a buffer solution (first reagent).
- Example 2 A buffer solution (first reagent) and a phospholipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that the latex particles were changed to LotB.
- Example 3 A buffer solution (first reagent) and a phospholipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that the latex particles were changed to LotC.
- Example 4 A buffer solution (first reagent) and a phospholipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that the latex particles were changed to LotD.
- Example 1 A buffer solution (first reagent) and a phospholipid antigen-sensitized latex reagent (second reagent) were prepared in the same manner as in Example 1 except that the latex particles were changed to LotE.
- an insoluble carrier used for a highly reactive antiphospholipid antibody measurement reagent it is possible to provide an antiphospholipid antibody measurement reagent and a method of measuring an antiphospholipid antibody.
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Abstract
Description
本発明者らは、鋭意検討した結果、親水性部分の少ないリン脂質を不溶性担体に吸着させる場合、同じ粒子径でゼータ電位の異なる不溶性担体を比較すると、ゼータ電位の低い不溶性担体を用いた方が、より反応性の高い、即ち、高感度の抗リン脂質抗体測定試薬が得られることを見出し、本発明を完成させるに至った。
具体的には、本発明は、抗リン脂質抗体測定試薬に用いる不溶性担体であって、20mmol/Lのリン酸ナトリウム水溶液(pH7.4)において固形分濃度が0.1%となるように懸濁した場合のゼータ電位が-45mV未満である抗リン脂質抗体測定試薬に用いる不溶性担体である。
また、本発明は、前記不溶性担体を用いた抗リン脂質抗体測定試薬、及び、前記不溶性担体を用いた抗リン脂質抗体の測定方法である。
以下に本発明を詳述する。
上記有機高分子粉末は、天然高分子粉末、合成高分子粉末が挙げられる。
上記合成高分子粉末は特に限定されず、例えば、ポリスチレン、スチレン-スルホン酸(塩)共重合体、スチレン-(メタ)アクリル酸共重合体、アクリロニトリル-ブタジエン-スチレン共重合体、塩化ビニル-(メタ)アクリル酸エステル共重合体、酢酸ビニル-(メタ)アクリル酸エステル共重合体等が挙げられる。
なかでも、合成高分子微粒子が水媒体中に均一に分散されたラテックス粒子であることが好適である。
上記フェニル基を有する重合性単量体と上記フェニル基及びスルホン酸基を有する重合性単量体とを共重合させる方法としては従来公知の方法を用いることができ、例えば、溶媒として水が仕込まれた反応容器内に上記フェニル基を有する重合性単量体、上記フェニル基とスルホン酸基を有する重合性単量体、重合開始剤、及び、必要に応じて乳化剤を添加し、窒素雰囲気下で攪拌する方法等が挙げられる。
なお、上記ラテックス粒子表面のスルホン酸基量は、電気伝導度滴定法(Journal of Colloid and Interface Sciences.49(3)425,1974)により求めることができ、この値を、得られた粒子径から算出される粒子の総表面積で除することにより、単位面積当たりのスルホン酸基量を算出することができる。
上記過硫酸塩類は特に限定されないが、過硫酸カリウム、過硫酸ナトリウム、過硫酸アンモニウム等が好適である。
上記重合開始剤の配合量は特に限定されないが、通常は重合性単量体量に対して0.01~1重量%の範囲である。
上記乳化剤の配合量は特に限定されないが、重合後の後処理工程により除去することを考慮すれば、フェニル基を有する重合性単量体に対して、好ましい上限は1重量%、より好ましい上限は0.5重量%、更に好ましい上限は0.02重量%である。上記乳化剤の配合量の好ましい下限は0.01重量%である。
なお、本明細書において(メタ)アクリル酸とは、アクリル酸又はメタクリル酸を意味する。
なお、上記粒子径のCV値(%)は、下記式により算出することができる。
粒子径のCV値(%)=粒子径の標準偏差/平均粒子径×100
上記リン脂質抗原は特に限定されないが、例えば、カルジオリピン、ホスファチジルコリン、及び、コレステロールからなるリン脂質抗原が好ましい。
上記カルジオリピンは、ウシの心臓から精製したものを用いることが好ましいが、化学的に合成されていてもよい。
上記ホスファチジルコリンは、ニワトリの卵黄から精製されたものを用いることが好ましいが、ホスファチジルコリンの含有量が60~80%であるレシチンを用いてもよい。また、ウシ心臓や大豆等から抽出されたものや、化学的に合成されたものでもよい。
上記コレステロールは、動物由来であってもよいし、化学的に合成されていてもよい。
上記緩衝液は、上記リン脂質抗原を担持したラテックス粒子を分散又は懸濁させる役割を有する。
上記緩衝液は特に限定されず、例えば、リン酸緩衝液、グリシン緩衝液、トリス塩緩衝液、グッド緩衝液等が挙げられる。
上記緩衝液のpHは特に限定されないが、好ましい下限は5.5、好ましい上限は8.5であり、より好ましい下限は6.5である。
上記水溶性高分子は特に限定されず、例えば、プルラン、ポリビニルピロリドン等が挙げられる。
なお、上記測定を行う際の光の波長は、300~900nmが好適である。
なお、凝集の度合いの観察には、目視による方法以外に、凝集状態をビデオカメラ等で撮影し、画像処理を施す方法を用いることも可能である。
(ラテックス粒子の作製)
攪拌機、還流用冷却器、温度検出器、窒素導入管及びジャケットを備えたガラス製反応容器(容量2L)に、蒸留水1100g、スチレン180g、スチレンスルホン酸ナトリウム0.04g、及び、蒸留水26gに過硫酸カリウム0.8gを溶解した水溶液を仕込み、容器内を窒素ガスで置換した後、70℃で攪拌しながら48時間重合した。
重合終了後、上記溶液をろ紙にてろ過処理し、ラテックス粒子を取り出した。得られたラテックス粒子の粒子径、表面のスルホン酸基量及びゼータ電位を下記の方法により測定した。
透過型電子顕微鏡装置(日本電子社製、「JEM-1010型」)を用いて10000倍の倍率でラテックス粒子を撮影し、最低100個以上の粒子について画像解析することにより粒子径を測定した。得られた平均粒子径は0.4μmであった。
ラテックス粒子をセロファンチューブ透析膜にて48時間、精製水で透析し残存単量体を除去した。この粒子を乾燥重量で10gになるように4ツ口ガラス容器に採取後、蒸留水で150mLになるように希釈しスターラーチップを用い攪拌した。これを溶液Aとした。
次に、電位差電気伝導度滴定処理装置(京都電子工業社製、「AT-310」)の付属装置ATB-310電動ビュレットに、0.01N水酸化ナトリウム(和光純薬工業社製)をセットし、更に導電率電極を溶液Aに浸し、窒素導入管、脱気管及びpH電極を設定した。そして、0.01N水酸化ナトリウムを滴下(0.05mLを150秒~500秒の範囲で測定するスルホン酸基量により調整)し、電位差電気伝導度滴定処理装置(京都電子工業社製、「AT-310」)を用いた伝導度の変化量から当量点を測定し、目的とするスルホン酸基量を算出した。得られたスルホン酸基量は0.28μmol/m2であった。
ラテックス粒子を20mmol/Lのリン酸ナトリウム水溶液(pH7.4)において固形分濃度が0.1%となるように調整し、これをゼータ電位測定用サンプルとした。
次に、ゼータ電位測定装置(Malvern Instruments Ltd.社製、「Zetasizer Nano ZEN3600」)にて、ゼータ電位測定用キャピラリーセルに測定サンプル750μLを分注し、測定温度37℃でゼータ電位を測定した。得られたゼータ電位は-74mVであった。
スチレンスルホン酸ナトリウムの配合量を0.08gとしたこと以外はLotAと同様にしてラテックス粒子を作製した。LotAと同様の方法で評価を行い、得られたラテックス粒子の平均粒子径は0.3μm、スルホン酸基量は0.23μmol/m2、ゼータ電位は-77mVであった。
蒸留水の配合量を1020g、スチレンスルホン酸ナトリウムの配合量を0.25gとし、蒸留水26gに過硫酸カリウム0.8gを溶解した水溶液の代わりに蒸留水13gに過硫酸カリウム0.4gを溶解した水溶液を用い、更に、0.1mol/Lリン酸水素二カリウム80gを添加したこと以外はLotAと同様にしてラテックス粒子を作製した。LotAと同様の方法で評価を行い、得られたラテックス粒子の平均粒子径は0.3μm、スルホン酸基量は0.16μmol/m2、ゼータ電位は-88mVであった。
蒸留水の配合量を1020g、スチレンスルホン酸ナトリウムの配合量を0.20gとし、蒸留水26gに過硫酸カリウム0.8gを溶解した水溶液の代わりに蒸留水16gに過硫酸カリウム0.6gを溶解した水溶液を用い、更に、0.1mol/Lリン酸水素二カリウム80gを添加したこと以外はLotAと同様にしてラテックス粒子を作製した。LotAと同様の方法で評価を行い、得られたラテックス粒子の平均粒子径は0.4μm、スルホン酸基量は0.18μmol/m2、ゼータ電位は-86mVであった。
スチレンスルホン酸ナトリウムの配合量を0.01gとし、蒸留水26gに過硫酸カリウム0.8gを溶解した水溶液の代わりに蒸留水4gに過硫酸カリウム0.1gを溶解した水溶液を用いたこと以外はLotAと同様にしてラテックス粒子を作製した。LotAと同様の方法で評価を行い、得られたラテックス粒子の平均粒子径は0.4μm、スルホン酸基量は0.06μmol/m2、ゼータ電位は-29mVであった。
(1)緩衝液(第1試薬)の調製
1.2(W/V)%のプルラン(林原社製)、1.0(W/V)%のウシ血清アルブミン(BSA)を含有する25mmol/Lのリン酸緩衝液(pH6.5)100mLに、塩化ナトリウム0.9gとアジ化ナトリウム0.1gを添加し、緩衝液(第1試薬)とした。
カルジオリピンのエタノール溶液(5mg/mL、シグマ社製)2mL、精製レシチン(ナカライテスク社製)のエタノール溶液(10mg/mL)10mL及びコレステロール(ナカライテスク社製)のエタノール溶液(10mg/mL)3mLを混合し、リン脂質抗原液を得た。
ラテックス粒子(LotA)100μLに、得られたリン脂質抗原液250μLを添加し、そのまま37℃で緩やかに2時間攪拌した。次に5(W/V)%濃度でBSAを含む100mmol/Lのリン酸緩衝液(pH6.5)3mLを添加し、更に37℃で1時間攪拌した。15000rpm、4℃で30分間遠心分離し、上澄みを除き、沈殿したラテックス粒子を1(W/V)%濃度でBSAを含む100mmol/Lのリン酸緩衝液(pH6.5)2mLに再び懸濁した。この操作を2回繰り返し、ラテックス粒子を洗浄し、最後にEDTA・4Naを10mmol/L、及び、塩化コリンを500mmol/L含む100mmol/Lのリン酸緩衝液(pH6.5)10mLに懸濁させてリン脂質抗原感作ラテックス試薬(第2試薬)とした。
ラテックス粒子をLotBに変更したこと以外は実施例1と同様にして、緩衝液(第1試薬)、リン脂質抗原感作ラテックス試薬(第2試薬)を調製した。
ラテックス粒子をLotCに変更したこと以外は実施例1と同様にして、緩衝液(第1試薬)、リン脂質抗原感作ラテックス試薬(第2試薬)を調製した。
ラテックス粒子をLotDに変更したこと以外は実施例1と同様にして、緩衝液(第1試薬)、リン脂質抗原感作ラテックス試薬(第2試薬)を調製した。
ラテックス粒子をLotEに変更したこと以外は実施例1と同様にして、緩衝液(第1試薬)、リン脂質抗原感作ラテックス試薬(第2試薬)を調製した。
実施例及び比較例で得られた緩衝液(第1試薬)、リン脂質抗原感作ラテックス試薬(第2試薬)について、以下の方法で評価した。
市販のRPR標準血清8.0R.U.(積水メディカル社製)20μLに、緩衝液(第1試薬)180μLを混合し、37℃で5分間保持した後、リン脂質抗原感作ラテックス試薬(第2試薬)60μLを添加攪拌し、測定波長700nmで添加後1分間及び5分間の吸光度変化を、日立7170形生化学自動分析機を用いて測定した。結果を表1及び図1に示す。
Claims (6)
- 抗リン脂質抗体測定試薬に用いる不溶性担体であって、
20mmol/Lのリン酸ナトリウム水溶液(pH7.4)において固形分濃度が0.1%となるように懸濁した場合のゼータ電位が-45mV未満である
ことを特徴とする抗リン脂質抗体測定試薬に用いる不溶性担体。 - 平均粒子径が、0.2μm~0.5μmであることを特徴とする請求項1記載の不溶性担体。
- 抗リン脂質抗体測定に用いる抗リン脂質抗体測定試薬であって、
リン脂質抗原を担持した不溶性担体と緩衝液とを含有し、
前記リン脂質抗原を担持する前の不溶性担体は、20mmol/Lのリン酸ナトリウム水溶液(pH7.4)において固形分濃度が0.1%となるように懸濁した場合のゼータ電位が-45mV未満である
ことを特徴とする抗リン脂質抗体測定試薬。 - 不溶性担体の平均粒子径が、0.2μm~0.5μmであることを特徴とする請求項3記載の抗リン脂質抗体測定試薬。
- リン脂質抗原を担持した不溶性担体と緩衝液とを含有する抗リン脂質抗体測定試薬と検体とを混合し、抗原抗体反応により凝集を生じさせる工程と、前記凝集の度合いを光学的に測定又は目視にて観察することにより、検体中の脂質抗体を測定する工程とを有する抗リン脂質抗体の測定方法であって、
リン脂質抗原を担持させる前の不溶性担体は、20mmol/Lのリン酸ナトリウム水溶液(pH7.4)において固形分濃度が0.1%となるように懸濁した場合のゼータ電位が-45mV未満である
ことを特徴とする抗リン脂質抗体の測定方法。 - 不溶性担体の平均粒子径が、0.2μm~0.5μmであることを特徴とする請求項5記載の抗リン脂質抗体の測定方法。
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EP20090826130 EP2352029A4 (en) | 2008-11-12 | 2009-11-12 | INSOLUBLE SUPPORT FOR USE IN A PHOSPHOLIPID ANTIBODY MEASUREMENT RUBBER, PHOSPHOLIPIDE ANTIBODY MEASUREMENT RUBBER AND PHOSPHOLIPID ANTIBODY MEASUREMENT METHOD |
CN200980144306.1A CN102203612B (zh) | 2008-11-12 | 2009-11-12 | 用于抗磷脂抗体测定试剂的不溶性载体、抗磷脂抗体测定试剂及抗磷脂抗体的测定方法 |
KR1020117008604A KR101690803B1 (ko) | 2008-11-12 | 2009-11-12 | 항인지질 항체 측정 시약에 사용하는 불용성 담체, 항인지질 항체 측정 시약, 및 항인지질 항체의 측정 방법 |
CA2741801A CA2741801C (en) | 2008-11-12 | 2009-11-12 | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
JP2010506751A JP4646270B2 (ja) | 2008-11-12 | 2009-11-12 | 抗リン脂質抗体測定試薬に用いる不溶性担体、抗リン脂質抗体測定試薬、及び、抗リン脂質抗体の測定方法 |
US13/128,718 US20110263048A1 (en) | 2008-11-12 | 2009-11-12 | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
HK12101022.6A HK1160680A1 (zh) | 2008-11-12 | 2012-02-03 | 用於抗磷脂抗體測定試劑的不溶性載體、抗磷脂抗體測定試劑及抗磷脂抗體的測定方法 |
US14/306,556 US20140295576A1 (en) | 2008-11-12 | 2014-06-17 | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
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US14/306,556 Division US20140295576A1 (en) | 2008-11-12 | 2014-06-17 | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
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