JP2010054516A - Immunoassay method preventing deviation between measurement data of serum and plasma - Google Patents
Immunoassay method preventing deviation between measurement data of serum and plasma Download PDFInfo
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- JP2010054516A JP2010054516A JP2009277755A JP2009277755A JP2010054516A JP 2010054516 A JP2010054516 A JP 2010054516A JP 2009277755 A JP2009277755 A JP 2009277755A JP 2009277755 A JP2009277755 A JP 2009277755A JP 2010054516 A JP2010054516 A JP 2010054516A
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- 238000003018 immunoassay Methods 0.000 title claims abstract description 36
- 238000005259 measurement Methods 0.000 title abstract description 30
- 210000002966 serum Anatomy 0.000 title abstract description 22
- 239000002738 chelating agent Substances 0.000 claims abstract description 70
- 150000002736 metal compounds Chemical class 0.000 claims abstract description 47
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 21
- 102000008946 Fibrinogen Human genes 0.000 claims abstract description 15
- 108010049003 Fibrinogen Proteins 0.000 claims abstract description 15
- 229940012952 fibrinogen Drugs 0.000 claims abstract description 15
- 239000004094 surface-active agent Substances 0.000 claims abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 5
- 239000002245 particle Substances 0.000 claims description 44
- 239000000872 buffer Substances 0.000 claims description 38
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 27
- 239000000725 suspension Substances 0.000 claims description 24
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 23
- 239000000203 mixture Substances 0.000 claims description 21
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 17
- 239000004816 latex Substances 0.000 claims description 16
- 229920000126 latex Polymers 0.000 claims description 16
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 230000004520 agglutination Effects 0.000 claims description 6
- 159000000007 calcium salts Chemical class 0.000 claims description 6
- 159000000003 magnesium salts Chemical class 0.000 claims description 6
- 235000006408 oxalic acid Nutrition 0.000 claims description 6
- IWTIBPIVCKUAHK-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)amino]propanoic acid Chemical compound OC(=O)CCN(CCC(O)=O)CCC(O)=O IWTIBPIVCKUAHK-UHFFFAOYSA-N 0.000 claims description 5
- 239000013522 chelant Substances 0.000 claims description 5
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical compound OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 238000003556 assay Methods 0.000 abstract 1
- 230000001900 immune effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 50
- 210000002381 plasma Anatomy 0.000 description 40
- 239000000427 antigen Substances 0.000 description 26
- 102000036639 antigens Human genes 0.000 description 26
- 108091007433 antigens Proteins 0.000 description 26
- 210000004369 blood Anatomy 0.000 description 22
- 239000008280 blood Substances 0.000 description 22
- 238000004220 aggregation Methods 0.000 description 7
- 239000012491 analyte Substances 0.000 description 7
- 230000002776 aggregation Effects 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000000691 measurement method Methods 0.000 description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 4
- 229920000053 polysorbate 80 Polymers 0.000 description 4
- 230000001235 sensitizing effect Effects 0.000 description 4
- 239000006173 Good's buffer Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- -1 collodion Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- ALWXETURCOIGIZ-UHFFFAOYSA-N 1-nitropropylbenzene Chemical compound CCC([N+]([O-])=O)C1=CC=CC=C1 ALWXETURCOIGIZ-UHFFFAOYSA-N 0.000 description 1
- AMBZRUJVCSJZSX-UHFFFAOYSA-N 4-imino-3-oxobutanoic acid Chemical compound OC(=O)CC(=O)C=N AMBZRUJVCSJZSX-UHFFFAOYSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- CBOCVOKPQGJKKJ-UHFFFAOYSA-L Calcium formate Chemical compound [Ca+2].[O-]C=O.[O-]C=O CBOCVOKPQGJKKJ-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000978776 Senegalia senegal Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 1
- 239000001639 calcium acetate Substances 0.000 description 1
- 235000011092 calcium acetate Nutrition 0.000 description 1
- 229960005147 calcium acetate Drugs 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- WUKWITHWXAAZEY-UHFFFAOYSA-L calcium difluoride Chemical compound [F-].[F-].[Ca+2] WUKWITHWXAAZEY-UHFFFAOYSA-L 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 229910001634 calcium fluoride Inorganic materials 0.000 description 1
- 229940095626 calcium fluoride Drugs 0.000 description 1
- 239000004281 calcium formate Substances 0.000 description 1
- 229940044172 calcium formate Drugs 0.000 description 1
- 235000019255 calcium formate Nutrition 0.000 description 1
- 239000004227 calcium gluconate Substances 0.000 description 1
- 235000013927 calcium gluconate Nutrition 0.000 description 1
- 229960004494 calcium gluconate Drugs 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 239000000920 calcium hydroxide Substances 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
- BCFSVSISUGYRMF-UHFFFAOYSA-N calcium;dioxido(dioxo)chromium;dihydrate Chemical compound O.O.[Ca+2].[O-][Cr]([O-])(=O)=O BCFSVSISUGYRMF-UHFFFAOYSA-N 0.000 description 1
- BIOOACNPATUQFW-UHFFFAOYSA-N calcium;dioxido(dioxo)molybdenum Chemical compound [Ca+2].[O-][Mo]([O-])(=O)=O BIOOACNPATUQFW-UHFFFAOYSA-N 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- MHJAJDCZWVHCPF-UHFFFAOYSA-L dimagnesium phosphate Chemical compound [Mg+2].OP([O-])([O-])=O MHJAJDCZWVHCPF-UHFFFAOYSA-L 0.000 description 1
- 229910000395 dimagnesium phosphate Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940095529 heparin calcium Drugs 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 239000011654 magnesium acetate Substances 0.000 description 1
- 235000011285 magnesium acetate Nutrition 0.000 description 1
- 229940069446 magnesium acetate Drugs 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- ARIXXGBZIGZIKW-UHFFFAOYSA-L magnesium dioxidophosphanium Chemical compound [Mg++].[O-][PH2]=O.[O-][PH2]=O ARIXXGBZIGZIKW-UHFFFAOYSA-L 0.000 description 1
- ORUIBWPALBXDOA-UHFFFAOYSA-L magnesium fluoride Chemical compound [F-].[F-].[Mg+2] ORUIBWPALBXDOA-UHFFFAOYSA-L 0.000 description 1
- 229910001635 magnesium fluoride Inorganic materials 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 229910001381 magnesium hypophosphite Inorganic materials 0.000 description 1
- BLQJIBCZHWBKSL-UHFFFAOYSA-L magnesium iodide Chemical compound [Mg+2].[I-].[I-] BLQJIBCZHWBKSL-UHFFFAOYSA-L 0.000 description 1
- 229910001641 magnesium iodide Inorganic materials 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920002102 polyvinyl toluene Polymers 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000003352 sequestering agent Substances 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- PBYZMCDFOULPGH-UHFFFAOYSA-N tungstate Chemical compound [O-][W]([O-])(=O)=O PBYZMCDFOULPGH-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
本発明は、抗原抗体凝集反応を光学的に抗原または抗体を測定する免疫測定法に関する。具体的には、フィブリノーゲンの影響により生じる血清被検試料と血漿被検試料における抗原または抗体の測定値の乖離を消失させ得る免疫測定法に関する。 The present invention relates to an immunoassay method for optically measuring an antigen or antibody in an antigen-antibody aggregation reaction. Specifically, the present invention relates to an immunoassay method capable of eliminating the difference between the measured values of antigens or antibodies in a serum test sample and a plasma test sample caused by fibrinogen.
抗原抗体凝集反応を光学的に抗原または抗体を測定する免疫測定法は、血清、血漿等のヒトから採取した生体成分を被検試料としてその中の微量成分を検出することにより、疾病等の生体の異常を診断する臨床検査法であり、自動分析装置を用いて簡単に定量できることから広く用いられている。
また、試料としては病院検査等で外来患者や緊急検査として迅速性から血漿が用いられている。
An immunoassay method for optically measuring an antigen-antibody or antigen-antigen-aggregation reaction is performed by detecting a trace component in a biological sample collected from a human, such as serum or plasma, and detecting a biological component such as a disease. It is a clinical test method for diagnosing abnormalities in the blood and is widely used because it can be easily quantified using an automatic analyzer.
In addition, as a sample, plasma is used for outpatients and emergency tests in hospital examinations and the like because of rapidity.
免疫測定法では、一般的に補体の影響を回避するために補体活性化経路に関与するCa2+、Mg2+と結合し得るエチレンジアミン四酢酸（以下EDTA）を含む免疫試薬が広く用いられている（非特許文献１を参照）。しかし、このEDTAが起因となり、血漿試料を測定した場合に不溶化物質が発生し、光学的測定に正の誤差を与え、同一ヒトより採血された血清試料の測定結果に対し乖離を生じる場合があった。 In immunoassays, immunoreagents containing ethylenediaminetetraacetic acid (EDTA) that can bind to Ca 2+ and Mg 2+ involved in the complement activation pathway are widely used to avoid the effects of complement. (See Non-Patent Document 1). However, due to this EDTA, insolubilized substances are generated when plasma samples are measured, giving positive errors to optical measurements, and there may be discrepancies in the measurement results of serum samples collected from the same human. It was.
そのため、これらEDTAを含む免疫測定法においては、血漿試料を用いた場合に誤った測定結果を与えるという問題があり、同一ヒトより採血された血清試料の測定値との誤差が生じない免疫試薬測定法が望まれていた。 For this reason, immunoassays involving these EDTAs have the problem of giving erroneous measurement results when plasma samples are used, and immunoreagent measurements that do not cause an error from the measured values of serum samples collected from the same human. The law was desired.
本発明の目的は、特定の特性を有するキレート剤および金属化合物を含む免疫測定試薬を用いることで、血漿試料と同一ヒトより採血された血清試料の測定値との誤差が生じない免疫試薬測定法を提供することである。 An object of the present invention is to provide an immunoreagent measurement method that does not cause an error between a plasma sample and a measurement value of a serum sample collected from the same human by using an immunoassay reagent containing a chelating agent and a metal compound having specific characteristics. Is to provide.
本願発明者らは血漿試料の正の測定誤差原因について調査した結果、血清中には無く、血漿中に存在するフィブリノーゲンが試薬中のEDTAによって不溶化し、光学的測定に影響を与え、測定に誤差を生じることを突き止めた。更に脂肪酸を疎水基に有する界面活性剤等の存在で誤差が助長されることもわかった。 As a result of investigating the cause of the positive measurement error of the plasma sample, the inventors of the present application found that fibrinogen present in the plasma, which is not in serum, was insolubilized by EDTA in the reagent, affecting optical measurement, and error in measurement. I found out that Furthermore, it was also found that errors are promoted by the presence of a surfactant having a fatty acid in a hydrophobic group.
そこで、免疫測定試薬に含まれるキレート剤について検討した結果、キレート剤の配位数が３以下の安定度定数の低いキレート剤を含む免疫測定法で血漿試料を測定すると、同一ヒトより採血された血清試料の測定値との誤差が生じないことがわかった。 Accordingly, as a result of examining chelating agents contained in immunoassay reagents, blood samples were collected from the same human when a plasma sample was measured by an immunoassay containing a chelating agent having a coordination number of the chelating agent of 3 or less and a low stability constant. It was found that there was no error from the measured value of the serum sample.
また、EDTAを含む採血管を用いた血漿試料から反応系に微量なＥＤＴＡが混入することで上記と同様な現象が起こることも確認された。これら回避方法も検討し、金属化合物を添加することで、EDTAをマスキングし、回避することもわかった。 It was also confirmed that the same phenomenon as described above occurred when a trace amount of EDTA was mixed into a reaction system from a plasma sample using a blood collection tube containing EDTA. We also studied these avoidance methods and found that adding a metal compound masks and avoids EDTA.
血漿試料中のフィブリノーゲンの不溶化機序については不明であるが、配位数が３以下のキレート剤の使用によりフィブリノーゲンの不溶化を抑え、採血管から混入するEDTAを金属化合物の添加でマスキングすることにより、血漿試料を誤差無く測定することが可能となった。 The mechanism of insolubilization of fibrinogen in plasma samples is unknown, but by using a chelating agent with a coordination number of 3 or less to suppress insolubilization of fibrinogen and masking EDTA mixed from blood collection tubes by adding metal compounds It became possible to measure plasma samples without error.
すなわち、本発明は、血漿試料をフィブリノーゲンの影響を受けずに測定する場合に、特定の特性を有するキレート剤を使用することを特徴とする方法を提供するものである。さらに、本発明は、EDTA採血管を用いて採血した被験試料中に含まれるEDTAの影響を低減するために免疫測定法において、金属化合物を使用することを特徴とする方法を提供するものである。 That is, the present invention provides a method characterized by using a chelating agent having specific characteristics when a plasma sample is measured without being affected by fibrinogen. Furthermore, the present invention provides a method characterized by using a metal compound in an immunoassay to reduce the effect of EDTA contained in a test sample collected using an EDTA blood collection tube. .
さらに、詳細には本発明は以下の通りである。
（１）血漿中の被検成分を抗原抗体反応により光学的に測定する免疫測定法において、反応系にキレート剤および／または金属化合物を添加することを含む、血漿中のフィブリノーゲンの不溶化による測定値への影響を回避する方法、
（２）免疫測定法が凝集反応を利用した方法である、（１）の方法、
（３）キレート剤が配位数３個以下のものであり、且つフィブリノーゲンの不溶化による測定値への影響を回避し得るものである（１）または（２）の方法、
（４）クエン酸およびクエン酸化合物、シュウ酸およびシュウ酸化合物、イミノ二酢酸、ニトリロトリスメチレンホスホン酸三ナトリウム塩、ニトリロ三プロピオン酸、およびこれらのキレート剤の類似化合物からなる群から選択されるキレート剤の１種類以上を添加する、（３）の方法、
（５）添加されるキレート剤の最終濃度が0.01〜0.1mol/Lである（１）から（４）のいずれかの方法、
（６）金属化合物が、カルシウム塩またはマグネシウム塩である（１）または（２）に記載の方法、
（７）添加される金属化合物の最終濃度が0.001〜0.05mol/Lである（１）、（２）または（６）の方法、
（８）血漿中の被検成分を抗原抗体反応により光学的に測定する免疫測定方法において血漿中のフィブリノーゲンの不溶化による測定値への影響を回避するための、キレート剤および／または金属化合物を含む緩衝液組成物、
（９）クエン酸およびクエン酸化合物、シュウ酸およびシュウ酸化合物、イミノ二酢酸、ニトリロトリスメチレンホスホン酸三ナトリウム塩、ニトリロ三プロピオン酸、およびこれらのキレート剤の類似化合物からなる群から選択されるキレート剤の１種類以上を含む（８）の緩衝液組成物、
（１０）キレート剤の濃度が0.01〜0.1mol/Lである（８）または（９）の緩衝液組成物、
（１１）金属化合物が、カルシウム塩またはマグネシウム塩である（８）の緩衝液組成物、
（１２）金属化合物の濃度が0.001〜0.05mol/Lである（１０）または（１１）の緩衝液組成物、および
（１３）抗原または抗体感作粒子を含む（８）〜（１２）のいずれかの緩衝液組成物。
Further, in detail, the present invention is as follows.
(1) In an immunoassay method in which a test component in plasma is optically measured by an antigen-antibody reaction, a measurement value by insolubilization of fibrinogen in plasma, including adding a chelating agent and / or a metal compound to the reaction system How to avoid impact on the
(2) The method according to (1), wherein the immunoassay is a method utilizing an agglutination reaction,
(3) The method according to (1) or (2), wherein the chelating agent has a coordination number of 3 or less, and the influence on the measured value due to insolubilization of fibrinogen can be avoided,
(4) selected from the group consisting of citric acid and citric acid compounds, oxalic acid and oxalic acid compounds, iminodiacetic acid, nitrilotrismethylenephosphonic acid trisodium salt, nitrilotripropionic acid, and analogs of these chelating agents Adding one or more of chelating agents, the method of (3),
(5) The method according to any one of (1) to (4), wherein the final concentration of the added chelating agent is 0.01 to 0.1 mol / L.
(6) The method according to (1) or (2), wherein the metal compound is a calcium salt or a magnesium salt,
(7) The method of (1), (2) or (6), wherein the final concentration of the added metal compound is 0.001 to 0.05 mol / L,
(8) A chelating agent and / or a metal compound is included in an immunoassay method for optically measuring a test component in plasma by an antigen-antibody reaction in order to avoid influence on measurement values due to insolubilization of fibrinogen in plasma. Buffer composition,
(9) selected from the group consisting of citric acid and citric acid compounds, oxalic acid and oxalic acid compounds, iminodiacetic acid, nitrilotrismethylenephosphonic acid trisodium salt, nitrilotripropionic acid, and analogs of these chelating agents (8) the buffer composition comprising one or more chelating agents,
(10) The buffer solution composition according to (8) or (9), wherein the concentration of the chelating agent is 0.01 to 0.1 mol / L,
(11) The buffer composition according to (8), wherein the metal compound is a calcium salt or a magnesium salt,
(12) Any of (8) to (12) comprising a buffer composition of (10) or (11) having a metal compound concentration of 0.001 to 0.05 mol / L, and (13) an antigen- or antibody-sensitized particle Such a buffer composition.
実施例に示したように、測定系に特定の特性を有するキレート剤を添加する本発明の免疫測定法により、血清検体と血漿検体における測定値の乖離を回避することができる。また、測定系に金属化合物を添加する本発明の免疫測定法により、EDTA採血管由来のEDTAの影響による血清検体と血漿検体における測定値の乖離を回避することができる。さらに測定系に上記キレート剤と金属化合物を添加することにより、血清検体と血漿検体における測定値の乖離をより十分に回避することができる。 As shown in the Examples, it is possible to avoid a difference in measured values between a serum sample and a plasma sample by the immunoassay method of the present invention in which a chelating agent having specific characteristics is added to the measurement system. Further, by the immunoassay method of the present invention in which a metal compound is added to the measurement system, it is possible to avoid the difference between the measured values in the serum sample and the plasma sample due to the influence of EDTA derived from the EDTA blood collection tube. Furthermore, by adding the chelating agent and the metal compound to the measurement system, it is possible to more sufficiently avoid the difference between the measured values of the serum sample and the plasma sample.
本発明は、血漿中の被検成分を抗原抗体反応により光学的に測定する免疫測定法において、反応系にキレート剤および／または金属化合物を添加することを含む、血漿中のフィブリノーゲンの不溶化による測定値への影響を回避する方法であり、また本発明は、血漿中のフィブリノーゲンの不溶化による測定値への影響を回避するために、反応系にキレート剤および／または金属化合物を添加することを含む、血漿中の被検成分を抗原抗体反応により光学的に測定する免疫測定方法である。 The present invention relates to an immunoassay method in which a test component in plasma is optically measured by an antigen-antibody reaction, comprising adding a chelating agent and / or a metal compound to the reaction system, and measuring by insolubilization of fibrinogen in plasma. The present invention includes adding a chelating agent and / or a metal compound to the reaction system in order to avoid the influence on the measurement value due to insolubilization of fibrinogen in plasma. This is an immunoassay method for optically measuring a test component in plasma by an antigen-antibody reaction.
本発明が対象とする免疫測定法は、抗原抗体反応により形成された抗原と抗体の結合物を光学的に測定する方法である。例えば、抗原抗体反応により生じた抗原または抗体を結合させた粒子の凝集反応を用いた方法があり、粒子に抗原または抗体を感作すなわち結合させ、被検試料中の抗体または抗原と粒子上の抗原または抗体が結合し、粒子が凝集体を形成し、濁度が変化する。粒子としてはラテックス粒子、ベントナイト、コロジオン、カオリン、固定羊赤血球、ゼラチン、金コロイド、炭素粒子等を使用することができるが、ラテックス粒子を使用するのが好ましい。ラテックス粒子としては、例えば、ポリスチレンラテックス粒子、スチレン-ブタジエン共重合体ラテックス粒子、ポリビニルトルエンラテックス粒子等を使用することができるが、ポリスチレンラテックス粒子を使用するのが好ましい。また、抗原抗体反応を光学的に検出するエンザイムイムノアッセイおよび蛍光イムノアッセイも本発明が対象とする免疫測定法である。例えば、抗原または抗体をマイクロタイタープレートに固相化するELISAや、抗原または抗体をビーズ等に固相化する不均一法ならびに結合した抗原および抗体複合体と遊離の抗体または抗原の分離操作（B/F分離）を行わない均一系測定法も本発明が対象とする免疫測定法である。後者の均一測定法として、例えば非競合均一エンザイムイムノアッセイやEMIT（登録商標）等の競合均一エンザイムイムノアッセイ等がある（P.TIJSSEN著、「生化学実験法11 エンザイムイムノアッセイ」、第１版、東京化学同人、1989年11月15日、p.14-17）。 The immunoassay targeted by the present invention is a method for optically measuring a conjugate of an antigen and an antibody formed by an antigen-antibody reaction. For example, there is a method using an agglutination reaction of an antigen produced by an antigen-antibody reaction or a particle to which an antibody is bound. The antigen or antibody is sensitized or bound to the particle, and the antibody or antigen in the test sample and the particle Antigens or antibodies bind, the particles form aggregates, and the turbidity changes. As particles, latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes, gelatin, colloidal gold, carbon particles and the like can be used, but it is preferable to use latex particles. As the latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles, and the like can be used, and polystyrene latex particles are preferably used. In addition, an enzyme immunoassay and a fluorescence immunoassay for optically detecting an antigen-antibody reaction are also immunoassays targeted by the present invention. For example, ELISA for immobilizing an antigen or antibody on a microtiter plate, a heterogeneous method for immobilizing an antigen or antibody on a bead or the like, and an operation for separating a bound antigen and antibody complex from a free antibody or antigen (B The homogeneous measurement method without (/ F separation) is also an immunoassay method targeted by the present invention. Examples of the latter uniform measurement method include non-competitive homogeneous enzyme immunoassay and competitive homogeneous enzyme immunoassay such as EMIT (registered trademark) (P. TIJSSEN, "Biochemical Experimental Method 11 Enzyme Immunoassay", 1st edition, Tokyo Chemical Co., Ltd.) Doujin, November 15, 1989, p.14-17).
本発明の免疫測定法における測定対象、すなわち被検成分は特定のものに限定されず、血中に存在する抗原または抗体ならばいずれでもよい。
本発明の方法に供される被検試料としては、血清、血漿、血液（全血）等の体液やその希釈物を挙げることができる。
The measurement target in the immunoassay method of the present invention, that is, the test component is not limited to a specific one, and any antigen or antibody present in blood may be used.
Examples of the test sample to be used in the method of the present invention include body fluids such as serum, plasma, blood (whole blood), and dilutions thereof.
例えば、凝集法において抗体または抗原を担体に感作する方法は、特に限定されず公知の方法に従えばよい。例えば、担体に物理的に吸着させてもよいし、化学的に結合させてもよい。より具体的には、例えば、抗体または抗原と担体とを混和した後、30〜37℃で1〜2時間加温振盪することにより、抗体を担体に感作させることができる。担体に感作する抗体または抗原の量は、使用する担体の粒径に応じて適宜設定することができる。抗体または抗原を担体に感作した後、担体表面上の未感作部分をウシ血清アルブミン、ヒト血清アルブミン、ウサギ血清アルブミン、卵白アルブミン等でブロッキングするのが好ましい。抗体または抗原を感作した担体は被検試料と反応させる時まで媒体分散液として保持しておくのが好ましい。この際、媒体としては、例えば、リン酸緩衝液、グリシン緩衝液等を使用することができる。媒体中には、必要に応じてウシ血清アルブミン、ゼラチン、アラビアゴム等を添加してもよい。このように調製した抗体または抗原感作担体を被検試料と反応させ、凝集の有無またはその程度により感作させた抗体または抗原と被検試料中の抗原または抗体との反応性を判別し、被検試料中の抗原または抗体を検出することができる。 For example, the method for sensitizing an antibody or antigen to a carrier in the aggregation method is not particularly limited and may be a known method. For example, it may be physically adsorbed on a carrier or chemically bonded. More specifically, for example, after mixing an antibody or antigen and a carrier, the antibody can be sensitized to the carrier by heating and shaking at 30 to 37 ° C. for 1 to 2 hours. The amount of antibody or antigen sensitized to the carrier can be appropriately set according to the particle size of the carrier used. After sensitizing the antibody or antigen to the carrier, the non-sensitized portion on the carrier surface is preferably blocked with bovine serum albumin, human serum albumin, rabbit serum albumin, ovalbumin or the like. The carrier sensitized with the antibody or antigen is preferably retained as a medium dispersion until it is reacted with the test sample. At this time, as the medium, for example, a phosphate buffer, a glycine buffer, or the like can be used. Bovine serum albumin, gelatin, gum arabic and the like may be added to the medium as necessary. The antibody or antigen-sensitized carrier thus prepared is reacted with a test sample, and the reactivity of the antibody or antigen sensitized with the presence or absence or the degree of aggregation is determined with the antigen or antibody in the test sample, An antigen or antibody in a test sample can be detected.
感作粒子を用いた凝集法における検出は、適当な反応容器中で被検試料数μL〜数十μLに生理食塩水または適当な緩衝液数十μL〜数百μLを添加し混和し、数分間インキュベーションを行い、次いで抗体または抗原を結合させた感作粒子を数十μL〜数百μL添加し、数分間インキュベーションを行う。次いで、適当な測定波長（例えば、570nm）で吸光度を測定することにより、抗原抗体反応により生じた感作粒子の凝集による濁度を測定することができる。また、反応試料数μLに緩衝液に浮遊させた感作粒子溶液数十μL〜数百μLを添加してもよい。緩衝液としては、pH5.0〜10の適当な緩衝液、例えば、リン酸緩衝液、ホウ酸緩衝液、トリス緩衝液等が挙げられる。この際、感作粒子と被検体中の抗原または抗体との非特異的結合を抑制するために、緩衝液にTritonX-100、Tween20、Tween80等の適当な界面活性剤を添加してもよい。凝集度の測定は、例えば三菱化学株式会社のLPIA-S500ラテックス凝集全自動測定器、ロシュ・ダイアグノスティック・システムズ社のCOBAS FARA装置及びCOBAS MIRA装置、及び日立製作所の日立7070分析装置等を用いて行うことができる。反応はスライドグラス上で行ってもよく、この場合凝集度は目視により判定してもよい。 Detection in the agglutination method using sensitized particles is performed by adding physiological saline or appropriate buffer solution of several tens of μL to several hundred μL to several μL to several tens of μL in a suitable reaction container, and mixing. Incubate for minutes, then add tens to hundreds of μL of sensitized particles to which antibody or antigen is bound, and incubate for several minutes. Next, by measuring the absorbance at an appropriate measurement wavelength (for example, 570 nm), turbidity due to aggregation of sensitized particles generated by the antigen-antibody reaction can be measured. Alternatively, several tens of μL to several hundred μL of the sensitized particle solution suspended in the buffer may be added to several μL of the reaction sample. Examples of the buffer include an appropriate buffer having a pH of 5.0 to 10, such as a phosphate buffer, a borate buffer, and a Tris buffer. At this time, an appropriate surfactant such as Triton X-100, Tween 20, or Tween 80 may be added to the buffer in order to suppress nonspecific binding between the sensitized particles and the antigen or antibody in the specimen. For example, the LPIA-S500 latex agglutination fully automatic measuring instrument manufactured by Mitsubishi Chemical Corporation, the COBAS FARA apparatus and the COBAS MIRA apparatus manufactured by Roche Diagnostics Systems, the Hitachi 7070 analyzer manufactured by Hitachi, Ltd. Can be done. The reaction may be carried out on a slide glass. In this case, the degree of aggregation may be determined visually.
本発明においては、最初に被検試料に添加する生理食塩水もしくは緩衝液、または感作粒子を浮遊させた緩衝液にキレート剤および／もしくは金属化合物を添加すればよい。また、最初に金属化合物のみを含む緩衝液と被検試料を混合し、被検試料中のEDTA採血管に由来するEDTAの配位座をマスキングし、その後にキレート剤含有緩衝液またはキレート剤含有感作粒子浮遊液を添加することによりキレート剤を添加してもよい。 In the present invention, a chelating agent and / or a metal compound may be added to a physiological saline solution or a buffer solution added to a test sample first, or a buffer solution in which sensitized particles are suspended. First, a buffer solution containing only a metal compound and a test sample are mixed, the EDTA coordination site derived from the EDTA blood collection tube in the test sample is masked, and then a chelating agent-containing buffer or chelating agent is contained. A chelating agent may be added by adding a sensitized particle suspension.
本発明で用いるキレート剤は配位数（配位座の数）３個以下のキレート剤であり、さらによりキレート安定度定数の低いものが好ましい。ここでキレート安定度係数とはL.G.Sillen & A.E.Martell著"StabilityConstants of Metal Complexes"The Chemical Society,London(1964)、S.Chaberek & A.E.Martell著"Organic Sequestering Agents"Willey(1959)等により一般に知られている係数をいう。本発明で用いるキレート剤は、血漿中の抗原または抗体を抗原抗体反応により光学的に測定する方法において、血漿中のフィブリノーゲンの不溶化させ測定値に影響を及ぼさず、なおかつ補体を活性化して測定値に影響を及ぼさないキレート剤である。血漿中のフィブリノーゲンの不溶化させ測定値に影響を及ぼすかどうかは、例えばフィブリノーゲン100〜700mg/mLの溶液にキレート剤を添加し、570nmにおける吸光度が増加しないかどうかで評価できる。本発明において用いるキレート剤の具体例として、クエン酸およびクエン酸化合物、シュウ酸およびシュウ酸化合物、イミノ二酢酸、ニトリロトリスメチレンホスホン酸三ナトリウム塩、ニトリロ三プロピオン酸、これらキレート剤の類似化合物が挙げられる。試料および感作粒子反応時の最終濃度は、0.01mol/L〜0.1mol/Lの範囲である。 例えば、キレート剤の濃度は試料10μLにキレート剤を含む緩衝液を0.2mL添加し、感作粒子浮遊液を0.1mL添加する測定系において、緩衝液中で0.015〜0.15mol/Lの範囲であり、0.05〜0.1mol/Lが好ましい。本発明の測定系としては、最初に被検体とキレート剤含有緩衝液を混合し、次いで感作粒子浮遊液を添加して反応を行わせる測定系および被検体とキレート剤含有感作粒子浮遊液を混合する測定系があり、系に添加する被検体、キレート剤含有緩衝液および感作粒子浮遊液の容積は適宜設定可能であるが、キレート剤の最終的な濃度が上記範囲になるように緩衝液または感作粒子浮遊液に含有させればよい。 The chelating agent used in the present invention is a chelating agent having a coordination number (number of coordination sites) of 3 or less, and further preferably a chelating agent having a lower chelate stability constant. Here, chelate stability coefficient is generally known by LGSillen & AEMartell "Stability Constants of Metal Complexes" The Chemical Society, London (1964), S. Chaberek & AEMartell "Organic Sequestering Agents" Willey (1959), etc. Coefficient. The chelating agent used in the present invention is a method of optically measuring an antigen or antibody in plasma by an antigen-antibody reaction, in which fibrinogen in plasma is insolubilized and does not affect the measured value, and is measured by activating complement. It is a chelating agent that does not affect the value. Whether or not fibrinogen in plasma is insolubilized and affects the measured value can be evaluated by, for example, adding a chelating agent to a 100 to 700 mg / mL solution of fibrinogen and not increasing the absorbance at 570 nm. Specific examples of chelating agents used in the present invention include citric acid and citric acid compounds, oxalic acid and oxalic acid compounds, iminodiacetic acid, nitrilotrismethylenephosphonic acid trisodium salt, nitrilotripropionic acid, and similar compounds of these chelating agents. Can be mentioned. The final concentration during the sample and sensitized particle reaction ranges from 0.01 mol / L to 0.1 mol / L. For example, the concentration of the chelating agent is in the range of 0.015 to 0.15 mol / L in the buffer solution in the measurement system in which 0.2 mL of the buffer containing the chelating agent is added to 10 μL of the sample and 0.1 mL of the sensitized particle suspension is added. 0.05 to 0.1 mol / L is preferable. As the measurement system of the present invention, first, a sample and a chelating agent-containing buffer solution are mixed, and then a sensitized particle suspension is added to cause the reaction, and the analyte and the chelating agent-containing sensitized particle suspension The volume of the analyte, chelating agent-containing buffer and sensitized particle suspension added to the system can be set as appropriate, but the final concentration of the chelating agent should be in the above range. It may be contained in a buffer solution or a sensitized particle suspension.
本発明において用いる金属化合物はEDTAと結合しEDTAの配位座をマスキングし得るものであればよく限定されないが、カルシウム塩、マグネシウム塩等をあげることができる。カルシウム塩としては、例えば、塩化カルシウム、水酸化カルシウム、炭酸カルシウム、タングステン酸カルシウム、モリブデン酸カルシウム、酢酸カルシウム、フッ化カルシウム、クロム酸カルシウム、グルコン酸カルシウム、ギ酸カルシウム、乳酸カルシウム、リン酸水素カルシウム、リン酸カルシウム、ケイ酸カルシウム、チオシアン酸カルシウム、硫酸カルシウム、ヘパリンカルシウム等が挙げられ、マグネシウム塩としては、塩化マグネシウム、フッ化マグネシウム、硫酸マグネシウム、ステアリン酸マグネシウム、オクタデカン酸マグネシウム、酢酸マグネシウム、炭酸マグネシウム、水酸化マグネシウム、硝酸マグネシウム、リン酸水素マグネシウム、リン酸マグネシウム、ステアリン酸マグネシウム、ホスフィン酸マグネシウム、ヨウ化マグネシウム等が挙げられる。 The metal compound used in the present invention is not particularly limited as long as it can bind to EDTA and mask the coordination position of EDTA, but examples thereof include calcium salts and magnesium salts. Examples of calcium salts include calcium chloride, calcium hydroxide, calcium carbonate, calcium tungstate, calcium molybdate, calcium acetate, calcium fluoride, calcium chromate, calcium gluconate, calcium formate, calcium lactate, and calcium hydrogen phosphate. , Calcium phosphate, calcium silicate, calcium thiocyanate, calcium sulfate, heparin calcium, etc., magnesium salts include magnesium chloride, magnesium fluoride, magnesium sulfate, magnesium stearate, magnesium octadecanoate, magnesium acetate, magnesium carbonate, Magnesium hydroxide, magnesium nitrate, magnesium hydrogen phosphate, magnesium phosphate, magnesium stearate, magnesium phosphinate Arm, and magnesium iodide and the like.
試料および感作粒子反応時の最終濃度は、0.001mol/L〜0.05mol/Lの範囲である。本発明で用いる金属化合物の濃度は試料10μLに金属化合物を含む緩衝液を0.2mL添加し、感作粒子浮遊液を0.1mL添加する測定系において緩衝液中で0.0015〜0.075mol/Lの範囲であり、0.01〜0.05mol/Lが好ましい。本発明の測定系としては、最初に被検体と金属化合物含有緩衝液を混合し、次いで感作粒子浮遊液を添加して反応を行わせる測定系および被検体と金属粒子含有感作粒子浮遊液を混合する測定系があり、反応系に添加する被検体、金属化合物含有緩衝液および感作粒子浮遊液の容積は適宜設定可能であるが、凝集反応時の金属化合物の最終的な濃度が上記範囲になるように緩衝液または感作粒子浮遊液に含有させればよい。 The final concentration during the sample and sensitized particle reaction ranges from 0.001 mol / L to 0.05 mol / L. The concentration of the metal compound used in the present invention is in the range of 0.0015 to 0.075 mol / L in the buffer solution in the measurement system in which 0.2 mL of the buffer solution containing the metal compound is added to 10 μL of the sample and 0.1 mL of the sensitized particle suspension is added. Yes, 0.01 to 0.05 mol / L is preferable. The measurement system of the present invention includes a measurement system in which an analyte and a metal compound-containing buffer are first mixed, and then a sensitized particle suspension is added to cause a reaction, and the analyte and the metal particle-containing sensitized particle suspension. The volume of the analyte, the metal compound-containing buffer and the sensitized particle suspension added to the reaction system can be set as appropriate, but the final concentration of the metal compound during the agglutination reaction is What is necessary is just to make it contain in a buffer solution or a sensitized particle suspension so that it may become a range.
また、測定系にキレート剤と金属化合物の両方を添加する場合、最初に被検体と金属化合物含有緩衝液を混合し、次いでキレート剤含有緩衝液を添加し、最後に感作粒子浮遊液を添加する系、最初に被検体とキレート剤と金属化合物の両方を含有する緩衝液を混合し、次いで感作粒子浮遊液を添加する系、最初に被検体と金属化合物含有緩衝液を混合し、次いでキレート剤含有浮遊液を添加する系、ならびに被検体とキレート剤と金属化合物の両方を含有する感作粒子浮遊液を添加する系等がある。どの系においても、反応系に添加する被検体、金属化合物含有緩衝液、キレート剤含有緩衝液、キレート剤と金属化合物の両方を含有する緩衝液ならびに感作粒子浮遊液の容積は適宜設定可能であり、いずれの場合であっても、凝集反応時のキレート剤および金属化合物の最終的な濃度が上記範囲になるように緩衝液または感作粒子浮遊液に含有させればよい。尚、あらかじめ緩衝液または感作粒子浮遊液にキレート剤と金属化合物の両方を混ぜておく場合には、キレート剤と金属化合物が結合し得るが、EDTA採血管で採取した血液を被検体として用いる場合には、本発明で用いるキレート剤に結合した金属化合物は、該キレート剤から遊離してEDTAと結合するので問題にならず、また金属化合物の濃度をキレート剤の濃度より低く設定すれば、ヘパリン採血管等のEDTAを含まない採血管を用いて採取した血液を被検体として用いる場合でも、金属化合物と結合していないキレート剤が存在するので、本発明の効果を奏し得る。この場合の、キレート剤と金属化合物のモル濃度比はキレート剤添加濃度と同モル以下の金属化合物で添加であればよい。本発明は、キレート剤および／または金属化合物を含む緩衝液組成物をも包含する。さらに、抗原または抗体を感作した粒子を含む緩衝液組成物も本発明に包含される。該緩衝液組成物は液体の状態であってもよいし、乾燥状態であってもよく、後者の場合水によって復元し使用することができる。さらに、本発明は前記緩衝液組成物を含む免疫測定キットをも包含する。該免疫測定キットは、前記緩衝液組成物に抗原または抗体を感作した粒子が含まれない場合は、抗原または抗体を感作した粒子を別途含む、さらに陽性対照、陰性対照、説明書等を含んでいてもよい。 When adding both chelating agents and metal compounds to the measurement system, first mix the analyte and the metal compound-containing buffer, then add the chelating agent-containing buffer, and finally add the sensitized particle suspension. A system in which a sample, a buffer containing both a chelating agent and a metal compound are first mixed, then a sensitizing particle suspension is added, a sample is first mixed with a buffer containing a metal compound, and then There are a system for adding a chelating agent-containing suspension, and a system for adding a sensitizing particle suspension that contains both the analyte, the chelating agent, and a metal compound. In any system, the volume of the sample to be added to the reaction system, the metal compound-containing buffer, the chelating agent-containing buffer, the buffer containing both the chelating agent and the metal compound, and the sensitized particle suspension can be set as appropriate. In any case, it may be contained in the buffer solution or the sensitized particle suspension so that the final concentration of the chelating agent and the metal compound during the aggregation reaction is in the above range. In addition, when both a chelating agent and a metal compound are mixed in a buffer solution or a sensitized particle suspension in advance, the chelating agent and the metal compound may be bound, but blood collected with an EDTA blood collection tube is used as a specimen. In this case, the metal compound bound to the chelating agent used in the present invention does not matter because it is released from the chelating agent and binds to EDTA, and if the concentration of the metal compound is set lower than the concentration of the chelating agent, Even when blood collected using a blood collection tube that does not contain EDTA, such as a heparin blood collection tube, is used as a subject, the effect of the present invention can be achieved because a chelating agent that is not bound to a metal compound is present. In this case, the molar concentration ratio between the chelating agent and the metal compound may be added as a metal compound having the same or lower mole as the chelating agent addition concentration. The present invention also includes a buffer composition comprising a chelating agent and / or a metal compound. Furthermore, a buffer composition containing particles sensitized with an antigen or antibody is also encompassed by the present invention. The buffer composition may be in a liquid state or in a dry state, and in the latter case, it can be reconstituted with water and used. Furthermore, the present invention also includes an immunoassay kit containing the buffer composition. The immunoassay kit, when the antigen or antibody-sensitized particles are not included in the buffer composition, further include particles sensitized with the antigen or antibody. May be included.
本明細書は本願の優先権の基礎である日本国特許出願2002-333714号の明細書および/または図面に記載される内容を包含する。 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2002-333714, which is the basis of the priority of the present application.
以下、本発明を実施例に基づき具体的に説明するが、本発明は下記実施例に限定されるものではない。なお、下記例において、「％」は「重量％」を示す。
〔参考例〕
ＲＦ測定試薬を用いて、緩衝液のキレート剤の種類における、同一ヒトから採血されたリウマチ因子を含む血清試料と血漿試料の誤差を調べた。この操作は具体的に次のように行なった。
EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to the following Example. In the following examples, “%” indicates “wt%”.
[Reference example]
Using an RF measurement reagent, the error between a serum sample containing rheumatoid factor collected from the same human and a plasma sample in the type of chelating agent in the buffer solution was examined. Specifically, this operation was performed as follows.
0.1mol/L Good緩衝液 pH8.3、30％ Tween80、0.05mol/L キレート剤を含む緩衝液を調製し、試料10μLに緩衝液0.2mLを混和し、37℃、５分間反応させた後、ラテックス浮遊液0.1mLを添加、混合しさらに５分間反応させ、測定波長570nmにて測定した。 0.1mol / L Good buffer pH 8.3, 30% Tween80, 0.05mol / L Chelate containing buffer is prepared, and 0.2mL of buffer is mixed with 10μL of sample and reacted at 37 ° C for 5 minutes. Latex suspension (0.1 mL) was added, mixed, reacted for an additional 5 minutes, and measured at a measurement wavelength of 570 nm.
〔実施例１〕 キレート剤の添加
次の組成から成る５種類の試薬を調製した。
５種類の試薬は緩衝液中に含まれるキレート剤が異なり、それ以外の成分は全て同じとした。
＜共通成分＞
緩衝液
Good緩衝液、pH8 0.1mol/L
Tween80 30％
キレート剤 0.05mol/L
ラテックス浮遊液
RF−ラテックスＸ１「生研」ラテックス浮遊液（デンカ生研）
＜試薬＞
緩衝液に以下のキレート剤を添加して測定を行った。
従来測定法
エチレンジアミン四酢酸（ナカライテスク）
試薬１
クエン酸三ナトリウム（ナカライテスク）
試薬２
シュウ酸（ナカライテスク）
試薬３
ニトリロ三プロピオン酸（同仁化学）
試薬４
イミノ二酢酸（同仁化学）
キレート剤の配位数を表１に示す。
[Example 1] Addition of chelating agent Five types of reagents having the following compositions were prepared.
The five reagents were different in the chelating agent contained in the buffer, and all other components were the same.
<Common ingredients>
Buffer
Good buffer, pH 8 0.1 mol / L
Tween80 30%
Chelating agent 0.05mol / L
Latex suspension
RF-Latex X1 "Seiken" Latex suspension (Denka Seiken)
<Reagent>
Measurement was performed by adding the following chelating agent to the buffer solution.
Conventional measurement method Ethylenediaminetetraacetic acid (Nacalai Tesque)
Reagent 1
Trisodium citrate (Nacalai Tesque)
Reagent 2
Oxalic acid (Nacalai Tesque)
Reagent 3
Nitriro tripropionic acid (Dojin Chemical)
Reagent 4
Iminodiacetic acid (Dojindo)
Table 1 shows the coordination number of the chelating agent.
ＲＦ陰性健常者より、血清および血漿を採血したものを試料とし、従来免疫測定法および試薬１〜４にて測定を行い、血清試料と血漿試料の測定値の差を算出した。
測定値の差（IU/mL）＝血漿試料の測定値−血清試料の測定値
結果を表２に示す。
A sample obtained by collecting serum and plasma from a healthy RF-negative person was used as a sample, and measurement was performed using a conventional immunoassay and reagents 1 to 4, and the difference between the measured values of the serum sample and the plasma sample was calculated.
Difference in measured value (IU / mL) = measured value of plasma sample−measured value of serum sample Table 2 shows the results.
表２に示されるように、キレート剤の配位数が３以下の選択されたキレート剤を含む免疫測定法では血漿試料と血清試料との測定値に差がないことがわかる。
〔実施例２〕 金属化合物の添加
実施例１で用いた試薬１の緩衝液に塩化カルシウムを添加しないものと添加したものを調製し、測定を行った。
＜試薬＞
緩衝液
good緩衝液、pH8 0.1mol/L
Tween80 30%
クエン酸三ナトリウム 0.05mol/L
塩化カルシウム 0.01mol/L
ラテックス浮遊液
ＲＦ−ラテックスＸ１「生研」ラテックス浮遊液（デンカ生研）
試薬１−１
塩化カルシウム無添加
試薬１−２
塩化カルシウム添加
As shown in Table 2, it can be seen that there is no difference in the measured value between the plasma sample and the serum sample in the immunoassay containing the selected chelating agent having a coordination number of the chelating agent of 3 or less.
[Example 2] Addition of metal compound The buffer solution of Reagent 1 used in Example 1 with and without addition of calcium chloride was prepared and measured.
<Reagent>
Buffer
good buffer, pH8 0.1 mol / L
Tween80 30%
Trisodium citrate 0.05mol / L
Calcium chloride 0.01mol / L
Latex suspension RF-latex X1 "Seiken" Latex suspension (Denka Seiken)
Reagent 1-1
Reagent without calcium chloride 1-2
Calcium chloride added
ＲＦ陰性健常人より、血清およびEDTA-2Na採血管にて採血した血漿（以下：EDTA採血管血漿試料）、ヘパリン-Li採血管にて採血した血漿（以下：ヘパリン採血管血漿試料）を試料とし、試薬１−１および試薬１−２にて測定を行い、血清試料とEDTA採血管血漿試料の測定値の差を算出した。
測定値の差（IU/mL）＝EDTA採血管血漿試料の測定値−血清試料の測定値
結果を表３に示す。
Serum and plasma collected from EDTA-2Na blood collection tubes (hereinafter: EDTA blood collection plasma samples) and plasma collected from heparin-Li blood collection tubes (hereinafter: heparin blood collection plasma samples) from healthy RF-negative healthy people Then, measurement was performed with Reagent 1-1 and Reagent 1-2, and the difference between the measured values of the serum sample and the EDTA blood collection plasma sample was calculated.
Difference in measured value (IU / mL) = measured value of EDTA blood collection plasma sample−measured value of serum sample Table 3 shows the results.
表３に示されるように、金属化合物を含む免疫測定法ではEDTA採血管の影響を回避し、EDTA採血管血漿試料と血清試料に差がないことがわかる。 As shown in Table 3, it can be seen that the immunoassay containing a metal compound avoids the influence of the EDTA blood collection tube, and there is no difference between the EDTA blood collection blood plasma sample and the serum sample.
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
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| JP2009277755A Expired - Lifetime JP5189067B2 (en) | 2002-11-18 | 2009-12-07 | Immunoassay to prevent divergence of serum and plasma measurements |
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| JP2018119959A (en) * | 2017-01-25 | 2018-08-02 | 三洋化成工業株式会社 | Method for producing antigen/antibody-immobilized particle, method for producing immunoassay reagent, and immunoassay method |
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| JP5143046B2 (en) * | 2009-02-12 | 2013-02-13 | 富士フイルム株式会社 | Test substance measurement method and kit for carrying out the measurement method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61280566A (en) * | 1984-09-03 | 1986-12-11 | Hiroaki Sawai | Direct immunoassay of 2'-5'-oligoadenylic acid |
| JPH0682450A (en) * | 1992-09-04 | 1994-03-22 | Eiken Chem Co Ltd | Immunological measuring reagent |
| JPH0720128A (en) * | 1993-07-06 | 1995-01-24 | Tosoh Corp | Method for measuring trace component in organic sample |
| JP2000210380A (en) * | 1999-01-26 | 2000-08-02 | Nagase & Co Ltd | Blood-coagulation inhibitor and blood drawing container housing the same |
| WO2002003068A1 (en) * | 2000-06-30 | 2002-01-10 | Kyowa Medex Co.,Ltd. | Insoluble carrier particle nephelometric immunoassay reagent |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1226795A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized coagulation control products |
| US5817525A (en) * | 1995-05-19 | 1998-10-06 | Chiron Diagnostics Corporation | Stable protein solutions for diagnostics and method of making and using the same |
-
2003
- 2003-11-18 JP JP2004553191A patent/JP4592422B2/en not_active Expired - Lifetime
- 2003-11-18 WO PCT/JP2003/014654 patent/WO2004046723A1/en active Application Filing
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2009
- 2009-12-07 JP JP2009277755A patent/JP5189067B2/en not_active Expired - Lifetime
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61280566A (en) * | 1984-09-03 | 1986-12-11 | Hiroaki Sawai | Direct immunoassay of 2'-5'-oligoadenylic acid |
| JPH0682450A (en) * | 1992-09-04 | 1994-03-22 | Eiken Chem Co Ltd | Immunological measuring reagent |
| JPH0720128A (en) * | 1993-07-06 | 1995-01-24 | Tosoh Corp | Method for measuring trace component in organic sample |
| JP2000210380A (en) * | 1999-01-26 | 2000-08-02 | Nagase & Co Ltd | Blood-coagulation inhibitor and blood drawing container housing the same |
| WO2002003068A1 (en) * | 2000-06-30 | 2002-01-10 | Kyowa Medex Co.,Ltd. | Insoluble carrier particle nephelometric immunoassay reagent |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018119959A (en) * | 2017-01-25 | 2018-08-02 | 三洋化成工業株式会社 | Method for producing antigen/antibody-immobilized particle, method for producing immunoassay reagent, and immunoassay method |
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| JP5189067B2 (en) | 2013-04-24 |
| JPWO2004046723A1 (en) | 2006-03-16 |
| JP4592422B2 (en) | 2010-12-01 |
| WO2004046723A1 (en) | 2004-06-03 |
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