JP5676498B2 - Immunological measuring reagent and measuring method for suppressing non-specific reaction - Google Patents
Immunological measuring reagent and measuring method for suppressing non-specific reaction Download PDFInfo
- Publication number
- JP5676498B2 JP5676498B2 JP2012009761A JP2012009761A JP5676498B2 JP 5676498 B2 JP5676498 B2 JP 5676498B2 JP 2012009761 A JP2012009761 A JP 2012009761A JP 2012009761 A JP2012009761 A JP 2012009761A JP 5676498 B2 JP5676498 B2 JP 5676498B2
- Authority
- JP
- Japan
- Prior art keywords
- fatty acid
- reagent
- acid
- sample
- immunoassay
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 111
- 238000006243 chemical reaction Methods 0.000 title claims description 52
- 238000000034 method Methods 0.000 title claims description 41
- 230000001900 immune effect Effects 0.000 title claims description 25
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 122
- 229930195729 fatty acid Natural products 0.000 claims description 122
- 239000000194 fatty acid Substances 0.000 claims description 122
- 150000004665 fatty acids Chemical class 0.000 claims description 69
- -1 fatty acid salt Chemical class 0.000 claims description 62
- 239000004816 latex Substances 0.000 claims description 53
- 229920000126 latex Polymers 0.000 claims description 53
- 238000005259 measurement Methods 0.000 claims description 40
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 claims description 37
- 238000003018 immunoassay Methods 0.000 claims description 34
- 230000000087 stabilizing effect Effects 0.000 claims description 34
- 239000000126 substance Substances 0.000 claims description 25
- 239000000427 antigen Substances 0.000 claims description 20
- 102000036639 antigens Human genes 0.000 claims description 20
- 108091007433 antigens Proteins 0.000 claims description 20
- 206010029719 Nonspecific reaction Diseases 0.000 claims description 19
- 230000004520 agglutination Effects 0.000 claims description 14
- WWZKQHOCKIZLMA-UHFFFAOYSA-N Caprylic acid Natural products CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 claims description 11
- FBUKVWPVBMHYJY-UHFFFAOYSA-N nonanoic acid Chemical compound CCCCCCCCC(O)=O FBUKVWPVBMHYJY-UHFFFAOYSA-N 0.000 claims description 11
- TUNFSRHWOTWDNC-UHFFFAOYSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 claims description 10
- 108060003951 Immunoglobulin Proteins 0.000 claims description 9
- 102000018358 immunoglobulin Human genes 0.000 claims description 9
- 235000021355 Stearic acid Nutrition 0.000 claims description 8
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 8
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 claims description 7
- WWZKQHOCKIZLMA-UHFFFAOYSA-M octanoate Chemical compound CCCCCCCC([O-])=O WWZKQHOCKIZLMA-UHFFFAOYSA-M 0.000 claims description 6
- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 125000005473 octanoic acid group Chemical group 0.000 claims description 4
- POULHZVOKOAJMA-UHFFFAOYSA-M dodecanoate Chemical compound CCCCCCCCCCCC([O-])=O POULHZVOKOAJMA-UHFFFAOYSA-M 0.000 claims description 3
- QIQXTHQIDYTFRH-UHFFFAOYSA-M octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC([O-])=O QIQXTHQIDYTFRH-UHFFFAOYSA-M 0.000 claims description 3
- 239000000523 sample Substances 0.000 description 56
- 239000002245 particle Substances 0.000 description 47
- 230000000694 effects Effects 0.000 description 21
- 230000000903 blocking effect Effects 0.000 description 17
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 15
- 229940098773 bovine serum albumin Drugs 0.000 description 15
- 230000002401 inhibitory effect Effects 0.000 description 15
- 241000711549 Hepacivirus C Species 0.000 description 13
- 239000002981 blocking agent Substances 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 239000006249 magnetic particle Substances 0.000 description 11
- 239000000463 material Substances 0.000 description 11
- 238000000691 measurement method Methods 0.000 description 11
- BYKRNSHANADUFY-UHFFFAOYSA-M sodium octanoate Chemical compound [Na+].CCCCCCCC([O-])=O BYKRNSHANADUFY-UHFFFAOYSA-M 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000004094 surface-active agent Substances 0.000 description 8
- 239000013076 target substance Substances 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 7
- 238000000576 coating method Methods 0.000 description 7
- 229920001577 copolymer Polymers 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000004793 Polystyrene Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 229920002223 polystyrene Polymers 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 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 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 230000006641 stabilisation Effects 0.000 description 5
- 238000011105 stabilization Methods 0.000 description 5
- 230000001629 suppression Effects 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 108090000371 Esterases Proteins 0.000 description 4
- 239000007987 MES buffer Substances 0.000 description 4
- 229920001214 Polysorbate 60 Polymers 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
- 235000021240 caseins Nutrition 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- BTURAGWYSMTVOW-UHFFFAOYSA-M sodium dodecanoate Chemical compound [Na+].CCCCCCCCCCCC([O-])=O BTURAGWYSMTVOW-UHFFFAOYSA-M 0.000 description 4
- LTOCMXUTASYUOC-UHFFFAOYSA-M sodium;nonanoate Chemical compound [Na+].CCCCCCCCC([O-])=O LTOCMXUTASYUOC-UHFFFAOYSA-M 0.000 description 4
- JUQGWKYSEXPRGL-UHFFFAOYSA-M sodium;tetradecanoate Chemical compound [Na+].CCCCCCCCCCCCCC([O-])=O JUQGWKYSEXPRGL-UHFFFAOYSA-M 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 241001227713 Chiron Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108010013296 Sericins Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 230000036436 anti-hiv Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- UKMSUNONTOPOIO-UHFFFAOYSA-N docosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCC(O)=O UKMSUNONTOPOIO-UHFFFAOYSA-N 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N ferric oxide Chemical compound O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 description 2
- XMHIUKTWLZUKEX-UHFFFAOYSA-N hexacosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCCC(O)=O XMHIUKTWLZUKEX-UHFFFAOYSA-N 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- CBFCDTFDPHXCNY-UHFFFAOYSA-N icosane Chemical compound CCCCCCCCCCCCCCCCCCCC CBFCDTFDPHXCNY-UHFFFAOYSA-N 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 150000004668 long chain fatty acids Chemical class 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002736 nonionic surfactant Substances 0.000 description 2
- UTOPWMOLSKOLTQ-UHFFFAOYSA-N octacosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCCCCC(O)=O UTOPWMOLSKOLTQ-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 150000004666 short chain fatty acids Chemical class 0.000 description 2
- 229920001059 synthetic polymer Polymers 0.000 description 2
- 208000006379 syphilis Diseases 0.000 description 2
- VHOCUJPBKOZGJD-UHFFFAOYSA-N triacontanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC(O)=O VHOCUJPBKOZGJD-UHFFFAOYSA-N 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical class OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- UNSRRHDPHVZAHH-WYTUUNCASA-N (5e,8e,11e)-icosa-5,8,11-trienoic acid Chemical compound CCCCCCCC\C=C\C\C=C\C\C=C\CCCC(O)=O UNSRRHDPHVZAHH-WYTUUNCASA-N 0.000 description 1
- CUXYLFPMQMFGPL-UHFFFAOYSA-N (9Z,11E,13E)-9,11,13-Octadecatrienoic acid Natural products CCCCC=CC=CC=CCCCCCCCC(O)=O CUXYLFPMQMFGPL-UHFFFAOYSA-N 0.000 description 1
- XUJWOMMOEOHPFP-UHFFFAOYSA-N (all-Z)-8,11-Eicosadienoic acid Natural products CCCCCCCCC=CCC=CCCCCCCC(O)=O XUJWOMMOEOHPFP-UHFFFAOYSA-N 0.000 description 1
- GYSCBCSGKXNZRH-UHFFFAOYSA-N 1-benzothiophene-2-carboxamide Chemical compound C1=CC=C2SC(C(=O)N)=CC2=C1 GYSCBCSGKXNZRH-UHFFFAOYSA-N 0.000 description 1
- AWQSAIIDOMEEOD-UHFFFAOYSA-N 5,5-Dimethyl-4-(3-oxobutyl)dihydro-2(3H)-furanone Chemical compound CC(=O)CCC1CC(=O)OC1(C)C AWQSAIIDOMEEOD-UHFFFAOYSA-N 0.000 description 1
- UNSRRHDPHVZAHH-UHFFFAOYSA-N 6beta,11alpha-Dihydroxy-3alpha,5alpha-cyclopregnan-20-on Natural products CCCCCCCCC=CCC=CCC=CCCCC(O)=O UNSRRHDPHVZAHH-UHFFFAOYSA-N 0.000 description 1
- XUJWOMMOEOHPFP-OKLKQMLOSA-N 8,11-Eicosadienoic acid Chemical compound CCCCCCCC\C=C\C\C=C\CCCCCCC(O)=O XUJWOMMOEOHPFP-OKLKQMLOSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 235000021357 Behenic acid Nutrition 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000003154 D dimer Substances 0.000 description 1
- GHVNFZFCNZKVNT-UHFFFAOYSA-N Decanoic acid Natural products CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101001121392 Homo sapiens Otoraplin Proteins 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 235000021353 Lignoceric acid Nutrition 0.000 description 1
- CQXMAMUUWHYSIY-UHFFFAOYSA-N Lignoceric acid Natural products CCCCCCCCCCCCCCCCCCCCCCCC(=O)OCCC1=CC=C(O)C=C1 CQXMAMUUWHYSIY-UHFFFAOYSA-N 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- 229920002292 Nylon 6 Polymers 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 241000473945 Theria <moth genus> Species 0.000 description 1
- 241000589886 Treponema Species 0.000 description 1
- UWHZIFQPPBDJPM-FPLPWBNLSA-M Vaccenic acid Natural products CCCCCC\C=C/CCCCCCCCCC([O-])=O UWHZIFQPPBDJPM-FPLPWBNLSA-M 0.000 description 1
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 150000001342 alkaline earth metals Chemical class 0.000 description 1
- 150000005215 alkyl ethers Chemical class 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 125000005037 alkyl phenyl group Chemical group 0.000 description 1
- CUXYLFPMQMFGPL-SUTYWZMXSA-N all-trans-octadeca-9,11,13-trienoic acid Chemical compound CCCC\C=C\C=C\C=C\CCCCCCCC(O)=O CUXYLFPMQMFGPL-SUTYWZMXSA-N 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940053200 antiepileptics fatty acid derivative Drugs 0.000 description 1
- 229910001566 austenite Inorganic materials 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 229940116226 behenic acid Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- GONOPSZTUGRENK-UHFFFAOYSA-N benzyl(trichloro)silane Chemical compound Cl[Si](Cl)(Cl)CC1=CC=CC=C1 GONOPSZTUGRENK-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 238000005219 brazing Methods 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006757 chemical reactions by type Methods 0.000 description 1
- SQNNHEYXAJPPKH-UHFFFAOYSA-N chloroethene;prop-2-enoic acid Chemical compound ClC=C.OC(=O)C=C SQNNHEYXAJPPKH-UHFFFAOYSA-N 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000011161 development Methods 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
- KFEVDPWXEVUUMW-UHFFFAOYSA-N docosanoic acid Natural products CCCCCCCCCCCCCCCCCCCCCC(=O)OCCC1=CC=C(O)C=C1 KFEVDPWXEVUUMW-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-MDZDMXLPSA-N elaidic acid Chemical compound CCCCCCCC\C=C\CCCCCCCC(O)=O ZQPPMHVWECSIRJ-MDZDMXLPSA-N 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 108010052295 fibrin fragment D Proteins 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229940106780 human fibrinogen Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- YAQXGBBDJYBXKL-UHFFFAOYSA-N iron(2+);1,10-phenanthroline;dicyanide Chemical compound [Fe+2].N#[C-].N#[C-].C1=CN=C2C3=NC=CC=C3C=CC2=C1.C1=CN=C2C3=NC=CC=C3C=CC2=C1 YAQXGBBDJYBXKL-UHFFFAOYSA-N 0.000 description 1
- YOBAEOGBNPPUQV-UHFFFAOYSA-N iron;trihydrate Chemical compound O.O.O.[Fe].[Fe] YOBAEOGBNPPUQV-UHFFFAOYSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000008297 liquid dosage form Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N n-hexanoic acid Natural products CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- SECPZKHBENQXJG-BQYQJAHWSA-N palmitelaidic acid Chemical compound CCCCCC\C=C\CCCCCCCC(O)=O SECPZKHBENQXJG-BQYQJAHWSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229920003214 poly(methacrylonitrile) Polymers 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 235000010483 polyoxyethylene sorbitan monopalmitate Nutrition 0.000 description 1
- 239000000249 polyoxyethylene sorbitan monopalmitate Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 235000021391 short chain fatty acids Nutrition 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000003445 sucroses Chemical class 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- QZZGJDVWLFXDLK-UHFFFAOYSA-N tetracosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCCCC(O)=O QZZGJDVWLFXDLK-UHFFFAOYSA-N 0.000 description 1
- UWHZIFQPPBDJPM-BQYQJAHWSA-N trans-vaccenic acid Chemical compound CCCCCC\C=C\CCCCCCCCCC(O)=O UWHZIFQPPBDJPM-BQYQJAHWSA-N 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229910000859 α-Fe Inorganic materials 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Description
本発明は、非特異反応を抑制する免疫学的測定試薬及び測定方法に関する。 The present invention relates to an immunological measurement reagent and a measurement method that suppress nonspecific reactions.
臨床検査薬分野において、不溶性担体を使用する免疫学的測定法が多用されている。例えば、ラテックス粒子の凝集反応を利用したラテックス凝集免疫測定法などや、免疫複合体を形成した粒子をＢ／Ｆ分離することを利用したＥＬＩＳＡ（酵素免疫測定法）、ＣＬＥＩＡ（化学発光酵素免疫測定法）などが挙げられる。 In the field of clinical diagnostics, immunoassays using insoluble carriers are frequently used. For example, latex agglutination immunoassay using agglutination reaction of latex particles, ELISA (enzyme immunoassay) using separation of B / F of particles that form immune complexes, CLEIA (chemiluminescence enzyme immunoassay) Law).
ラテックス凝集免疫測定法は、抗原又は抗体を固相したラテックス粒子と試料中の測定対象物質とを反応させ、抗原抗体反応を介してラテックス粒子を凝集させ、凝集の程度を光学的に測定又は目視で観察することにより測定対象物質を測定する方法である。例えば、ＨＣＶ（Ｃ型肝炎ウイルス）特異的ヒト免疫グロブリンを測定対象物質とする場合、ＨＣＶ抗原を固相化したラテックス粒子にＨＣＶ特異的ヒト免疫グロブリンを添加すると該ラテックス粒子が凝集するため、その凝集の程度からＨＣＶ特異的ヒト免疫グロブリンの存在量を定量できる。 Latex agglutination immunoassay is a method in which latex particles solidified with an antigen or antibody are reacted with a substance to be measured in a sample, latex particles are aggregated via an antigen-antibody reaction, and the degree of aggregation is optically measured or visually observed. This is a method for measuring a substance to be measured by observing in FIG. For example, when HCV (hepatitis C virus) -specific human immunoglobulin is used as a substance to be measured, the addition of HCV-specific human immunoglobulin to latex particles on which HCV antigen is immobilized causes the latex particles to aggregate. The abundance of HCV-specific human immunoglobulin can be quantified from the degree of aggregation.
一方、ＥＬＩＳＡやＣＬＥＩＡの一例として磁性粒子を利用した免疫学的測定方法がある。これは、試料中の測定対象物質と特異的に反応する抗体又は抗原を固相化した磁性粒子と、試料中の測定対象物質とを抗原抗体反応させた後、Ｂ／Ｆ分離し、磁性粒子を洗浄した後に、該測定対象物質と特異的に反応する標識された抗原又は抗体を反応させ、当該標識を利用した発色又は発光を検出し、測定対象物質を測定する方法である。例えば、ＨＣＶ特異的ヒト免疫グロブリンを測定する場合は、ＨＣＶ抗原を固相化した磁性粒子と、ヒト免疫グロブリンとを抗原抗体反応させ、抗原固相化磁性粒子をＢ／Ｆ分離後洗浄し、標識抗体を反応させ、ＨＣＶ特異的ヒト免疫グロブリンの存在量を定量できる。 On the other hand, as an example of ELISA or CLEIA, there is an immunological measurement method using magnetic particles. This is because magnetic particles in which an antibody or antigen that specifically reacts with a measurement target substance in a sample is solid-phased and a measurement target substance in a sample are subjected to antigen-antibody reaction, and then B / F separation is performed. After washing, a labeled antigen or antibody that specifically reacts with the measurement target substance is reacted, and color development or luminescence using the label is detected to measure the measurement target substance. For example, when measuring HCV-specific human immunoglobulin, the magnetic particles on which HCV antigen is immobilized and human immunoglobulin are reacted with an antigen antibody, and the antigen-immobilized magnetic particles are washed after B / F separation, The abundance of HCV-specific human immunoglobulin can be quantified by reacting the labeled antibody.
生体由来の試料によっては、不溶性担体又は不溶性担体に担持した成分に吸着等する因子（非特異反応因子）が存在する場合がある。この場合、測定しようとする測定対象物質が試料中に存在しない場合であっても陽性の測定値を示し、真値と異なる測定値を示す場合がある。逆に本来の抗原抗体反応が阻害され、偽陰性となる場合もある。これら、測定対象物質に係る目的の抗原抗体反応とは異なる反応を非特異反応という（非特許文献２）。 Depending on the sample derived from a living body, an insoluble carrier or a factor (nonspecific reaction factor) that is adsorbed on a component carried on the insoluble carrier may exist. In this case, even when the measurement target substance to be measured does not exist in the sample, a positive measurement value may be shown and a measurement value different from the true value may be shown. Conversely, the original antigen-antibody reaction may be inhibited, resulting in false negatives. These reactions different from the target antigen-antibody reaction related to the measurement target substance are called non-specific reactions (Non-patent Document 2).
ラテックス凝集免疫測定法で生じる非特異反応には、非特異吸着物質が結合する対象により、少なくとも３つの類型が知られている。
第一は、ラテックス粒子自体に非特異吸着物質が結合する類型である。ラテックス粒子は通常、タンパク質で表面を固相被覆して用いる（特許文献１、非特許文献２）。しかし、被覆が十分でなかったり、被覆成分がラテックス粒子から剥がれたりする場合、ラテックス粒子の表面が露出し、試料由来の非特異吸着物質が当該表面へ吸着し、非特異的な凝集反応が生じる。前記第一の類型の非特異反応を回避する目的で、ウシ血清アルブミン（以下、ＢＳＡ）やカゼイン等によりラテックス粒子の表面を被覆する手段が多用されている。これにより、ラテックス粒子表面が被覆され、第一の類型の非特異反応を回避できる（特許文献１）。
There are at least three types of nonspecific reactions that occur in latex agglutination immunoassay depending on the target to which the nonspecifically adsorbed substance binds.
The first is a type in which a nonspecifically adsorbed substance binds to the latex particles themselves. Latex particles are usually used by solid-phase-coating the surface with protein (
しかしながら、ＢＳＡやカゼインには抗原性があるため、試料中にＢＳＡ等に反応する抗体が含まれると非特異凝集反応が生じる。これが第二の類型である（非特許文献３）。
また、ラテックス粒子には、測定対象物質に特異的に反応する抗体又は抗原を固相して使用するが、当該抗体又は抗原に対し、目的とする抗原抗体反応とは異なる抗原抗体反応が生じる場合がある。これが第三の類型である（非特許文献１、非特許文献２）。第三の類型は、ラテックス粒子に固相するタンパク質が、測定する試料の動物種と異なる動物種のタンパク質である場合に生じやすい。例えば、ラテックス粒子にマウス抗体を固相すると、ヒトの試料中のヒト抗マウス抗体（Ｈｕｍａｎ Ａｎｔｉ−Ｍｏｕｓｅ Ａｎｔｉｂｏｄｙ：ＨＡＭＡ）を含むと、非特異的な凝集を生じる（非特許文献１、非特許文献２）。この問題を解消するため、キメラ抗体やヒト化抗体が利用できることが報告されている（特許文献１）
However, since BSA and casein have antigenicity, nonspecific agglutination occurs when an antibody that reacts with BSA or the like is contained in the sample. This is the second type (Non-Patent Document 3).
The latex particles are used in solid phase with antibodies or antigens that react specifically with the substance to be measured. When antigen-antibody reactions that differ from the target antigen-antibody reactions occur against the antibodies or antigens. There is. This is the third type (
一方、Ｂ／Ｆ分離を利用した免疫学的測定方法においても、上記非特異反応の類型が同様に生じる。すなわち、例えば第一類型であれば、試料中の非特異吸着物質が磁粒子表面に吸着する場合に、それに対し標識抗体が反応し、検出される現象である。疾患の同定や治療方針の選択のため、カットオフ値を設けて陰性／陽性を判定する試薬において、磁性粒子表面に対する非特異の吸着反応が著しいと、陰性と判断されるべき試料が陽性と判定されるので非常に問題になる。 On the other hand, in the immunological measurement method using B / F separation, the above non-specific reaction type is similarly generated. That is, for example, in the first type, when a non-specifically adsorbed substance in a sample is adsorbed on the surface of a magnetic particle, this is a phenomenon in which the labeled antibody reacts and is detected. In the reagent for determining negative / positive by setting a cut-off value for disease identification and treatment policy selection, if the non-specific adsorption reaction on the magnetic particle surface is remarkable, the sample that should be judged as negative is judged as positive It will be very problematic.
粒子表面に対する非特異反応を回避する為、タンパク質等で粒子を被覆する手法が多用されている。目的とする抗原抗体反応に関与しないタンパク質をラテックス粒子や磁性粒子の表面に接触させる工程を経ることで、疎水的性質の強い粒子表面を被覆し非特異吸着物質の吸着を軽減するものである（以下、ブロッキングあるいは非特異抑制と言う）。ブロッキング剤としては、ＢＳＡ、熱変性させたＢＳＡ、アルカリ処理をしたＢＳＡ、カゼイン、スキムミルク、セリシン、ゼラチン、合成高分子ポリマー等が知られ、実際に多用されている（特許文献２）。 In order to avoid non-specific reactions on the particle surface, a method of coating particles with proteins or the like is frequently used. Through the process of bringing the protein not involved in the target antigen-antibody reaction into contact with the surface of latex particles or magnetic particles, the surface of particles with strong hydrophobic properties is coated to reduce adsorption of nonspecifically adsorbed substances ( Hereinafter referred to as blocking or non-specific suppression). As the blocking agent, BSA, heat-denatured BSA, alkali-treated BSA, casein, skim milk, sericin, gelatin, synthetic polymer, and the like are known, and are actually used frequently (Patent Document 2).
前記ブロッキング剤で被覆する場合、固相されたブロッキング剤に特異的に反応する抗体が非特異反応を生じる問題がある（第二類型）。例えば、ラテックス粒子のブロッキング剤としてＢＳＡを使用する場合、ヒト試料中の抗ＢＳＡ抗体が固相されたＢＳＡに結合し凝集する（非特許文献３）。また、スキムミルクはブロッキング作用が強いが、抗原抗体反応の阻害作用（抗原のマスク作用）もある点で問題である。ＢＳＡを変性してブロッキング剤に用いる技術もあるが、ＢＳＡの原料ロットが異なると調製条件が異なる場合があり、ロット管理の面で煩雑である。これら種々の課題が生じるため、前記第一の類型の非特異抑制対策としては、ブロッキング剤の使用は最良の非特異抑制手段とは言えず、より優れた手段が求められていた。 When coating with the blocking agent, there is a problem that an antibody that reacts specifically with the solid-phased blocking agent causes a non-specific reaction (second type). For example, when BSA is used as a blocking agent for latex particles, the anti-BSA antibody in the human sample binds and aggregates with the solid-phased BSA (Non-patent Document 3). In addition, skim milk has a strong blocking action, but is problematic in that it also has an antigen-antibody reaction inhibition action (antigen masking action). There is also a technique of modifying BSA and using it as a blocking agent, but preparation conditions may be different if the BSA raw material lot is different, which is complicated in terms of lot management. Because of these various problems, the use of a blocking agent is not the best non-specific suppression means as the first type of non-specific suppression measures, and more excellent means have been demanded.
この他に特許文献３には、粒子表面に対する非特異反応を回避する手段として、界面活性剤を添加し、非特異吸着物質の除去又は吸着阻害をし、非特異吸着物質の影響を緩和する方法が開示されている。ラテックス凝集免疫法では、試料中に含まれる複数のタンパク質が非特異反応の原因物質と考えられている。原因物質の一つとして試料血清、血漿中のマイクロフィブリン等の血液凝固因子があるが、界面活性剤を添加しても作用が弱く、多量の原因物質を除去するには充分な効果を示すものではなかった。また、多量の界面活性剤を添加すれば、再現性や特異性等他の試薬性能を劣化させる問題もある。 In addition to this, Patent Document 3 discloses a method for adding a surfactant as a means for avoiding a non-specific reaction on the particle surface, removing or inhibiting the adsorption of the non-specific adsorption substance, and mitigating the influence of the non-specific adsorption substance. Is disclosed. In the latex agglutination immunization method, it is considered that a plurality of proteins contained in a sample are causative substances of a nonspecific reaction. One of the causative substances is blood serum coagulation factors such as sample serum and plasma microfibrin, but even if a surfactant is added, the action is weak, and it is effective enough to remove a large amount of causative substances It wasn't. Further, if a large amount of surfactant is added, there is a problem that other reagent performances such as reproducibility and specificity are deteriorated.
その他に、特許文献４には、不溶性担体の表面素材と同じ素材からなる粒子又は担体を試料と接触させ、試料中の非特異吸着物質を吸収する手段が開示されている。当該粒子自体が光学的に測り込まれ検出されるため、高感度で正確な測定を要求される臨床検査試薬キットにおいて当該技術は最良とは言えない。すなわち、試薬に非特異吸着物質を吸収する粒子を添加しておくと、非特異吸着物質が当該粒子を凝集させる為、光学的に検出される程度の凝集体が形成され、当該凝集体が測定系を干渉し、正確な測定が妨げられるのである。 In addition, Patent Document 4 discloses a means for bringing particles or a carrier made of the same material as the surface material of an insoluble carrier into contact with a sample and absorbing nonspecifically adsorbed substances in the sample. Since the particles themselves are optically measured and detected, the technique is not the best for clinical test reagent kits that require high sensitivity and accurate measurement. In other words, if particles that absorb non-specifically adsorbed substances are added to the reagent, the nonspecifically adsorbed substances aggregate these particles, so that aggregates that are optically detected are formed and the aggregates are measured. Interfering with the system prevents accurate measurement.
特許文献５においては、疎水性物質を固定化した担体と試料を処理し、非特異的に結合する物質を除去する技術が開示されている。大型の自動分析機を用い、大量の試料をより早く測定することが求められている臨床検査業務の現状に鑑みると、自動分析機での測定前に測定者による用手法での試料処理が必要な状況は望まれず、特殊な前処理が必要となる臨床検査薬には最良とは言えない。従って、例えば、自動分析装置で利用できるような臨床検査薬の一般的な剤形、すなわち均一に分散された液状の剤形であることが望ましく、特許文献５の技術は当該剤形においては達成できない。
本発明者らの目的は、前記非特異反応の特に第一類型あるいは第二類型を抑制することができる新規の非特異抑制剤、免疫学的測定試薬及び測定方法を提供することである。例えば、粒子をブロッキング剤で被覆しなくとも非特異反応を抑制できる手段を提供することである。 An object of the present inventors is to provide a novel nonspecific inhibitor, immunoassay reagent and measurement method capable of suppressing the first type or second type of the nonspecific reaction. For example, it is to provide means that can suppress non-specific reactions without coating the particles with a blocking agent.
本発明者らは、不溶性担体を使用する免疫学的測定試薬において非特異反応の抑制方法を鋭意研究した結果、脂肪酸及び／又は脂肪酸塩が、ブロッキング剤で被覆しない不溶性担体に対しても非特異反応を強く抑制することを見出し、本発明を完成させた。 As a result of diligent research on a method for suppressing non-specific reaction in an immunoassay reagent using an insoluble carrier, the present inventors have found that fatty acids and / or fatty acid salts are non-specific even for insoluble carriers not coated with a blocking agent. The inventors have found that the reaction is strongly suppressed and completed the present invention.
すなわち、本発明者らは、定法に従って界面活性剤添加での非特異反応抑制効果を検証していたところ、偶然にポリオキシエチレンソルビタンモノパルミタート（Ｔｗｅｅｎ２０：polyoxyethylene sorbitan monopalmitate）へエステラーゼが混入し、驚くべきことにこのエステラーゼの混入したＴｗｅｅｎ２０溶液は非特異抑制効果が著しく増強していることに気付いた。
That is, the present inventors have verified the inhibitory effect of non-specific reaction by adding a surfactant according to a conventional method. By chance, esterase is mixed into polyoxyethylene sorbitan monopalmitate (Tween 20), Surprisingly, it was found that the
鋭意研究の結果、エステラーゼがＴｗｅｅｎ２０を分解し、分解産物の脂肪酸に強い非特異抑制効果があり、そして脂肪酸を溶解した試薬では、非特異反応が生じず、不溶性担体をブロッキング処理しなくとも非特異反応を抑制できるという驚くべき高い効果があることを見出し、本発明を完成させた。
As a result of diligent research, esterase decomposes
水溶液の試薬に脂溶性の成分を添加することは、不溶性成分の沈殿の発生等、試薬性能を劣化させると信じられており、これまで脂肪酸の存在下で抗原抗体反応を行うことは、避けられてきた。すなわち、不溶性成分が水溶液中に不均一に存在する状況となると、正確な測定結果を得られないため、試薬を設計する際には、親水性の成分を優先的に試み、添加剤として採用され、水難溶性の成分は積極的に避けられてきた現状である。従って、脂肪酸を臨床検査薬へ溶解させる発想は、当業者にとって思想上の障害がある。 Adding fat-soluble components to aqueous reagents is believed to degrade the reagent performance, such as the precipitation of insoluble components, and so far, antigen-antibody reactions in the presence of fatty acids are unavoidable. I came. In other words, when insoluble components are present unevenly in an aqueous solution, accurate measurement results cannot be obtained, so hydrophilic components are preferentially tried and used as additives when designing reagents. The poorly water-soluble component has been actively avoided. Therefore, the idea of dissolving a fatty acid in a clinical test drug has an ideological obstacle for those skilled in the art.
すなわち、本発明は、
［１］不溶性担体を含有する免疫学的測定試薬であって、脂肪酸及び／又は脂肪酸塩を含むことを特徴とする、免疫学的測定試薬、
［２］脂肪酸及び／又は脂肪酸塩の炭素数が４〜３０である、［１］の免疫学的測定試薬、
［３］免疫学的測定が、ラテックス凝集免疫測定法、あるいは、Ｂ／Ｆ分離を伴う免疫学的測定法である、［１］又は［２］の免疫学的測定試薬、
［４］安定化試薬及び反応試薬を含む免疫学的測定試薬であって、該安定化試薬及び／又は該反応試薬に脂肪酸及び／又は脂肪酸塩を含む、［１］〜［３］のいずれかの免疫学的測定試薬、
［５］前記脂肪酸及び／又は脂肪酸塩が、オクタン酸及び／又はオクタン酸塩、ノナン酸及び／又はノナン酸塩、ドデカン酸及び／又はドデカン酸塩、テトラデカン酸及び／又はテトラデカン酸塩、オクタデカン酸及び／又はオクタデカン酸塩から少なくとも一つ選ばれる、［１］〜［４］のいずれかの免疫学的測定試薬、
［６］前記脂肪酸及び／又は脂肪酸塩が、オクタン酸及び／又はオクタン酸塩である、［１］〜［５］のいずれかのの免疫学的測定試薬、
［７］測定対象物質がヒト免疫グロブリンである、［１］〜［６］のいずれかの免疫学的測定試薬、
［８］不溶性担体を使用する免疫学的測定方法であって、脂肪酸及び／又は脂肪酸塩を含むことを特徴とする、免疫学的測定方法、
［９］試料を安定化試薬と混合する第一の工程と、
次いで、測定対象物質に対する抗原又は抗体を担持させた不溶性担体を含む反応液と反応させる第二の工程とを含み、
脂肪酸及び／又は脂肪酸塩が、安定化試薬及び／又は反応液に溶解されている、免疫学的測定方法
に関する。
That is, the present invention
[1] An immunoassay reagent containing an insoluble carrier, which comprises a fatty acid and / or a fatty acid salt,
[2] The immunoassay reagent of [1], wherein the fatty acid and / or fatty acid salt has 4 to 30 carbon atoms,
[3] The immunological measurement reagent according to [1] or [2], wherein the immunological measurement is a latex agglutination immunoassay or an immunological measurement with B / F separation,
[4] Any one of [1] to [3], which is an immunoassay reagent containing a stabilizing reagent and a reaction reagent, wherein the stabilizing reagent and / or the reaction reagent contains a fatty acid and / or a fatty acid salt. An immunoassay reagent,
[5] The fatty acid and / or fatty acid salt is octanoic acid and / or octanoate, nonanoic acid and / or nonanoate, dodecanoic acid and / or dodecanoate, tetradecanoic acid and / or tetradecanoate, octadecanoic acid And / or at least one selected from octadecanoate, the immunoassay reagent according to any one of [1] to [4],
[6] The immunoassay reagent according to any one of [1] to [5], wherein the fatty acid and / or fatty acid salt is octanoic acid and / or octanoate.
[7] The immunological measurement reagent according to any one of [1] to [6], wherein the substance to be measured is human immunoglobulin,
[8] An immunological measurement method using an insoluble carrier, which comprises a fatty acid and / or a fatty acid salt,
[9] a first step of mixing the sample with a stabilizing reagent;
Next, a second step of reacting with a reaction solution containing an insoluble carrier carrying an antigen or antibody against the substance to be measured,
The present invention relates to an immunological measurement method in which a fatty acid and / or a fatty acid salt is dissolved in a stabilizing reagent and / or a reaction solution.
本発明によれば、不溶性担体を使用する免疫学的方法で生じる非特異反応を抑制し、高感度、高精度に試料中の測定対象物質を測定可能とする免疫学的測定試薬及び測定方法を提供することができる。更には、不溶性担体をブロッキング処理することなく非特異反応を抑制できる高い効果を有する。 According to the present invention, there are provided an immunological measurement reagent and a measurement method capable of suppressing a non-specific reaction caused by an immunological method using an insoluble carrier and measuring a measurement target substance in a sample with high sensitivity and high accuracy. Can be provided. Furthermore, it has a high effect of suppressing non-specific reaction without blocking the insoluble carrier.
本発明は、不溶性担体を用いる免疫学的測定試薬及び測定方法において、脂肪酸及び／または脂肪酸塩により非特異反応を抑制することが特徴である。
以下、本発明について更に詳細に説明する。
The present invention is characterized in that a nonspecific reaction is suppressed by a fatty acid and / or a fatty acid salt in an immunological measurement reagent and measurement method using an insoluble carrier.
Hereinafter, the present invention will be described in more detail.
本発明で用いる脂肪酸及び／又は脂肪酸塩は、非特異反応抑制効果が認められれば特に限定しないが、飽和又は不飽和、直鎖又は分岐鎖が挙げられる。 The fatty acid and / or fatty acid salt used in the present invention is not particularly limited as long as a non-specific reaction inhibitory effect is observed, and examples thereof include saturated or unsaturated, linear or branched chain.
飽和脂肪酸としては、例えば、ブタン酸、ペンタン酸、ヘキサン酸、ヘプタン酸、オクタン酸、ノナン酸、デカン酸、ドデカン酸、テトラデカン酸、ペンタデカン酸、ヘキサデカン酸、ヘプタデカン酸、オクタデカン酸、ノデカン酸、エイコサン酸、ドコサン酸、テトラコサン酸、ヘキサコサン酸、オクタコサン酸、トリアコンタン酸等が挙げられる。 Examples of saturated fatty acids include butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nodecanoic acid, eicosane Examples include acid, docosanoic acid, tetracosanoic acid, hexacosanoic acid, octacosanoic acid, triacontanoic acid and the like.
不飽和脂肪酸としては、例えば、９−ヘキサデセン酸、ｃｉｓ−９−オクタデセン酸、１１−オクタデセン酸、ｃｉｓ，ｃｉｓ−９，１２−オクタデカジエン酸、９，１２，１５−オクタデカントリエン酸、６，９，１２−オクタデカトリエン酸、９，１１，１３−オクタデカトリエン酸、８，１１−エイコサジエン酸、５，８，１１−エイコサトリエン酸、５，８，１１−エイコサテトラエン酸、ｃｉｓ−１５−テトラコサン酸等が挙げられる。 Examples of unsaturated fatty acids include 9-hexadecenoic acid, cis-9-octadecenoic acid, 11-octadecenoic acid, cis, cis-9,12-octadecadienoic acid, 9,12,15-octadecantrienoic acid, 6, 9,12-octadecatrienoic acid, 9,11,13-octadecatrienoic acid, 8,11-eicosadienoic acid, 5,8,11-eicosatrienoic acid, 5,8,11-eicosatetraenoic acid, Examples thereof include cis-15-tetracosanoic acid.
脂肪酸塩は上記脂肪酸の水酸基が置換されているものが挙げられるが、例えば、ナトリウム、カリウム等のアルカリ金属、カルシウム、マグネシウム等のアルカリ土類金属との塩を挙げることができる。 Examples of the fatty acid salt include those in which the hydroxyl group of the fatty acid is substituted, and examples thereof include salts with alkali metals such as sodium and potassium, and alkaline earth metals such as calcium and magnesium.
本発明で用いる脂肪酸及び／又は脂肪酸塩は、市販の調製された脂肪酸又は脂肪酸塩を用いてもよいし、脂肪酸の誘導体から酵素等で分解して調製してもよい。
脂肪酸の誘導体としては、例えば、一価又は多価アルコールとのエステル、リン脂質、及びこれらのエステルにエチレンオキサイドを付加した誘導体を挙げることができる。具体的には、例えば、上記脂肪酸のメチルエステル、エチルエステル、モノグリセリド、ジグリセリド、それらのエチレンオキサイド付加体、ポリグリセリンエステル、ソルビタンエステル、ショ糖エステル等が挙げられる。
脂肪酸は、前記脂肪酸誘導体を、例えば、エステラーゼやリパーゼ等の酵素により加水分解して得ることができる。または、測定試薬に前記脂肪酸誘導体と前記酵素とを添加することで、実質的に脂肪酸を添加するのと同等な効果を得るものでもよい。当業者であれば、公知の方法に従って、容易に実施することができる。
また、これらの脂肪酸、脂肪酸塩又はその誘導体は、２種以上を併用してもよい。
The fatty acid and / or fatty acid salt used in the present invention may be a commercially available fatty acid or fatty acid salt, or may be prepared by decomposing from a fatty acid derivative with an enzyme or the like.
Examples of fatty acid derivatives include esters with mono- or polyhydric alcohols, phospholipids, and derivatives obtained by adding ethylene oxide to these esters. Specific examples include methyl esters, ethyl esters, monoglycerides, diglycerides, ethylene oxide adducts thereof, polyglycerin esters, sorbitan esters, and sucrose esters of the above fatty acids.
The fatty acid can be obtained by hydrolyzing the fatty acid derivative with an enzyme such as esterase or lipase. Alternatively, the fatty acid derivative and the enzyme may be added to the measurement reagent to obtain an effect substantially equivalent to that of adding a fatty acid. A person skilled in the art can easily carry out according to a known method.
Moreover, these fatty acids, fatty acid salts, or derivatives thereof may be used in combination of two or more.
本発明で使用可能な脂肪酸及び／又は脂肪酸塩の濃度は、水溶液に溶解させることができる範囲であれば特に限定されない。当業者であれば、選択する脂肪酸及び／又は脂肪酸塩の種類により水溶液への溶解度が異なる点、測定する際の試料と脂肪酸含有試薬との比に応じ、測定条件毎に最適な添加濃度を容易に決定することができる。 The concentration of the fatty acid and / or fatty acid salt that can be used in the present invention is not particularly limited as long as it can be dissolved in an aqueous solution. A person skilled in the art can easily obtain the optimum concentration for each measurement condition according to the difference in solubility in aqueous solution depending on the type of fatty acid and / or fatty acid salt selected and the ratio of the sample to the fatty acid-containing reagent at the time of measurement. Can be determined.
例えば、オクタン酸ナトリウムを選択し、試料とオクタン酸ナトリウム含有試薬の液量比（試料の液量／オクタン酸ナトリウム含有組成物）を０．１６とする場合には（以下、本段落において同じ）、オクタン酸ナトリウム含有試薬におけるオクタン酸ナトリウム濃度が０．０５〜２．０重量％で効果が優れ、０．２〜２．０重量％であるとより効果が優れ、０．５〜２．０重量％であると更に効果が優れる。
また、ノナン酸ナトリウムの場合は、０．１〜１．５重量％で効果が優れ、０．５〜１．５重量％であるとより効果が優れる。
また、ドデカン酸の場合は、０．０５〜１．０重量％で効果が優れ、０．１〜１．０重量％であるとより効果が優れる。
また、ドデカン酸ナトリウムの場合は、０．０５〜１．０重量％で効果が優れ、０．２〜１．０重量％であるとより効果が優れ、０．５〜１．０重量％であると更に効果が優れる。
また、テトラデカン酸ナトリウムの場合は、０．０１〜０．５重量％で効果が優れ、０．０２〜０．５重量％であるとより効果が優れる。
また、オクタデカン酸の場合は、０．０１〜０．２重量％で効果が優れ、０．０５〜０．２重量％であるとより効果が優れる。
これらの濃度は、脂肪酸又は脂肪酸塩含有試薬（試料と当該試薬の液量比＝０．１６）中の脂肪酸又は脂肪酸塩の溶解濃度であり、試料と当該試薬とを混合した際の混合液中の脂肪酸又は脂肪酸塩の溶解濃度は、これらの濃度を１．１６で除することにより算出することができる。
For example, when sodium octoate is selected and the liquid volume ratio of the sample to the sodium octoate-containing reagent (the liquid volume of the sample / sodium octoate-containing composition) is 0.16 (hereinafter the same in this paragraph) The effect is excellent when the sodium octoate concentration in the sodium octoate-containing reagent is 0.05 to 2.0% by weight, and the effect is more excellent when it is 0.2 to 2.0% by weight. The effect is further excellent when the content is wt%.
In the case of sodium nonanoate, the effect is excellent at 0.1 to 1.5% by weight, and the effect is more excellent at 0.5 to 1.5% by weight.
In the case of dodecanoic acid, the effect is excellent at 0.05 to 1.0% by weight, and the effect is more excellent at 0.1 to 1.0% by weight.
In addition, in the case of sodium dodecanoate, the effect is excellent at 0.05 to 1.0% by weight, the effect is more excellent at 0.2 to 1.0% by weight, and 0.5 to 1.0% by weight. If present, the effect is further improved.
In the case of sodium tetradecanoate, the effect is excellent at 0.01 to 0.5% by weight, and the effect is more excellent at 0.02 to 0.5% by weight.
In the case of octadecanoic acid, the effect is excellent at 0.01 to 0.2% by weight, and the effect is more excellent at 0.05 to 0.2% by weight.
These concentrations are the dissolved concentrations of the fatty acid or fatty acid salt in the fatty acid or fatty acid salt-containing reagent (the liquid volume ratio of the sample and the reagent = 0.16), and in the mixed liquid when the sample and the reagent are mixed The dissolution concentration of the fatty acid or fatty acid salt can be calculated by dividing these concentrations by 1.16.
短鎖脂肪酸の場合、非特異抑制効果を奏するのに高濃度の添加が必要で、長鎖脂肪酸では低濃度での添加で済む傾向がある。
長鎖脂肪酸は、難溶性であること等で不具合を生じやすく、また、短鎖脂肪酸は、異臭が酷くて製品上は使いづらいことから、炭素数が４〜３０、好ましくは６〜２０、より好ましくは８〜１８の脂肪酸または脂肪酸塩が使用できる。
具体的には、オクタン酸及び／又はオクタン酸塩、ノナン酸及び／又はノナン酸塩、ドデカン酸及び／又はドデカン酸塩、テトラデカン酸及び／又はテトラデカン酸塩、オクタデカン酸及び／又はオクタデカン酸塩が挙げられる。特に好ましくは、オクタン酸及び／又はオクタン酸塩が使用できる。
In the case of a short-chain fatty acid, addition of a high concentration is necessary to produce a nonspecific inhibitory effect, and in the case of a long-chain fatty acid, there is a tendency that addition at a low concentration is sufficient.
Long chain fatty acids are prone to problems due to poor solubility and the like, and short chain fatty acids have a bad odor and are difficult to use on the product, so the number of carbon atoms is 4 to 30, preferably 6 to 20, and more. Preferably 8-18 fatty acids or fatty acid salts can be used.
Specifically, octanoic acid and / or octanoate, nonanoic acid and / or nonanoate, dodecanoic acid and / or dodecanoate, tetradecanoic acid and / or tetradecanoate, octadecanoic acid and / or octadecanoate are included. Can be mentioned. Particularly preferably, octanoic acid and / or octanoate can be used.
これらの脂肪酸は水溶液に直接加えてもよいが、分散性を改善するために、有機溶剤に脂肪酸を一旦分散、溶解させて添加してもよい。該有機溶剤としては、例えば、クロロホルム、メタノール、エタノール等が挙げられるが、好ましくはエタノールがよい。 These fatty acids may be added directly to the aqueous solution, but in order to improve dispersibility, the fatty acids may be once dispersed and dissolved in an organic solvent. Examples of the organic solvent include chloroform, methanol, ethanol and the like, and ethanol is preferable.
水溶液に溶解し難い脂肪酸の場合、界面活性剤を共存させることで溶解させることができる。該界面活性剤は、過度な検討を要さず、公知のものの中から適宜選択して使用することができるが、特に非イオン性界面活性剤が好ましい。前記非イオン性界面活性剤としては、例えば、ポリオキシエチレンアルキルエーテル、ソルビタン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレンアルキルフェニルエーテル、アルキルポリグリコシド、脂肪酸アルカノールアミド、ショ糖脂肪酸エステルを挙げることができる。好ましくはソルビタン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレンアルキルフェニルエーテル、より好ましくはソルビタン脂肪酸エステルまたはポリオキシエチレンソルビタン脂肪酸エステル、更に好ましくはポリオキシエチレンソルビタン脂肪酸エステル等が挙げられる。前記界面活性剤を、２種類以上を混合して使用してもよい。 In the case of a fatty acid that is difficult to dissolve in an aqueous solution, it can be dissolved by coexisting a surfactant. The surfactant does not require undue investigation and can be appropriately selected from known ones, and a nonionic surfactant is particularly preferable. Examples of the nonionic surfactant include polyoxyethylene alkyl ether, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl phenyl ether, alkyl polyglycoside, fatty acid alkanolamide, and sucrose fatty acid ester. be able to. Preferred examples include sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkylphenyl ethers, more preferably sorbitan fatty acid esters or polyoxyethylene sorbitan fatty acid esters, and still more preferably polyoxyethylene sorbitan fatty acid esters. Two or more kinds of the surfactants may be mixed and used.
前記界面活性剤は、本発明で使用可能な脂肪酸及び／又は脂肪酸塩を含む試薬に０．０１〜５重量％、好ましくは０．１〜３重量％となるように含有させるのがよい。 The surfactant is contained in a reagent containing a fatty acid and / or a fatty acid salt that can be used in the present invention in an amount of 0.01 to 5% by weight, preferably 0.1 to 3% by weight.
本発明で使用する脂肪酸及び／又は脂肪酸塩は、溶液中で溶解している状態の他、安定に分散又は乳濁している状態であっても発明の効果を奏するが、臨床検査薬としては、保存安定性や正確な測定が維持されるように、溶解している状態が最も望ましいことから、溶液中に溶解していることが好ましい。
当業者であれば、使用する形態や条件に合わせ、適宜、本発明で使用する脂肪酸及び脂肪酸塩を選択し、調製することができる。
The fatty acid and / or fatty acid salt used in the present invention has the effect of the invention even in a state of being stably dispersed or emulsified in addition to a state dissolved in a solution, Since the dissolved state is most desirable so that storage stability and accurate measurement are maintained, it is preferable to dissolve in a solution.
A person skilled in the art can appropriately select and prepare the fatty acid and fatty acid salt used in the present invention in accordance with the form and conditions to be used.
本発明の免疫学的測定方法としては、不溶性担体を使用する免疫学的測定方法であれば良く、ラテックス凝集免疫測定法などの免疫学的粒子凝集反応、Ｂ／Ｆ分離を行う粒子等の支持体を用いたエンザイムイムノアッセイ（ＥＬＩＳＡ法）、免疫比ろう法、酵素免疫測定法、蛍光免疫測定法、放射免疫測定法等が使用できる。また、競合法の原理に基づくＲＩＡ法やＥＬＩＳＡ法でも使用できる。 The immunological measurement method of the present invention may be any immunological measurement method using an insoluble carrier, and supports immunological particle agglutination reactions such as latex agglutination immunoassay, particles that perform B / F separation, etc. Enzyme immunoassay (ELISA method) using the body, immuno-ratio brazing method, enzyme immunoassay method, fluorescent immunoassay method, radioimmunoassay method and the like can be used. It can also be used in the RIA method and ELISA method based on the principle of the competitive method.
また、本発明の免疫学的測定方法は、１液もしくは２液以上から構成される免疫学的測定試薬を用いて実施することができる。 The immunological measurement method of the present invention can be carried out using an immunological measurement reagent composed of one liquid or two liquids or more.
１液から構成される場合、少なくとも免疫学的複合体を形成するための抗体あるいは抗原を担持させた支持体を含む反応試薬からなる。公知の方法に従い、試料を該試薬と反応させ、シグナルを検出する。本発明で用いる脂肪酸及び／又は脂肪酸塩は、試料と該試薬とが反応する前に、該試薬に添加されていると良いが、好ましくは、該試薬に添加された状態で供給することができる。 When it is composed of one solution, it comprises at least a reaction reagent including a support carrying an antibody or antigen for forming an immunological complex. In accordance with a known method, the sample is reacted with the reagent, and the signal is detected. The fatty acid and / or fatty acid salt used in the present invention may be added to the reagent before the sample and the reagent react with each other. Preferably, the fatty acid and / or fatty acid salt can be supplied in a state of being added to the reagent. .
２液以上から構成される場合、安定化試薬と少なくとも免疫学的複合体を形成するための抗体あるいは抗原を担持させた支持体を含む反応試薬からなる。安定化試薬は、試料を適当な濃度に希釈したり、前処理を行ったりする試薬で、公知の方法に従って調製することができる。公知の方法に従い、試料を安定化試薬と反応させ、その後、反応試薬と反応性、シグナルを検出する。本発明で用いる脂肪酸及び／又は脂肪酸塩は、試料と該反応試薬とが反応する前に、該安定化試薬及び／又は該反応試薬に添加されていると良いが、好ましくは、該安定化試薬及び／又は該反応試薬に添加された状態で供給することができる。 In the case of being composed of two or more liquids, it consists of a reaction reagent including a support on which an antibody or an antigen for forming an immunological complex at least with a stabilizing reagent is supported. The stabilizing reagent is a reagent for diluting a sample to an appropriate concentration or performing pretreatment, and can be prepared according to a known method. In accordance with a known method, the sample is reacted with the stabilizing reagent, and then the reactivity with the reaction reagent and the signal are detected. The fatty acid and / or fatty acid salt used in the present invention may be added to the stabilizing reagent and / or the reaction reagent before the sample and the reaction reagent react, but preferably the stabilizing reagent. And / or can be supplied in the state of being added to the reaction reagent.
例えば、自動測定装置を使用する臨床検査薬の場合には、測定原理、試薬を搭載する自動測定機の種類、測定機での試薬液量パラメーター等の観点から、その剤形を選択することができる。
一般にはラテックス凝集免疫測定法においては２液系からなる場合が多い。試料を希釈する安定化試薬と、ラテックス粒子が分散されたラテックス試薬とから構成される。本発明で用いる脂肪酸及び／又は脂肪酸塩は最終的に試料と混和される態様であればよく、安定化試薬に含有されていても良いし、ラテックス試薬に含有されていても良い。更に、前記試薬とは別に、脂肪酸及び／又は脂肪酸塩を含有する水溶液を用意し、測定前に安定化試薬に添加しても良いし、ラテックス試薬に添加しても良い。
Ｂ／Ｆ分離を行う磁性粒子を使用する免疫学的測定法においても、２から３液系で構成される場合が多いが、上記と同様に何れの構成試薬に本発明の脂肪酸及び／又は脂肪酸塩を含有あるいは添加させてもよい。
For example, in the case of a clinical test drug that uses an automatic measuring device, the dosage form can be selected from the viewpoints of the measurement principle, the type of automatic measuring machine equipped with the reagent, the reagent liquid volume parameter in the measuring machine, etc. it can.
In general, latex agglutination immunoassay often consists of two liquid systems. It is composed of a stabilizing reagent for diluting a sample and a latex reagent in which latex particles are dispersed. The fatty acid and / or fatty acid salt used in the present invention may be in an embodiment finally mixed with the sample, and may be contained in the stabilizing reagent or may be contained in the latex reagent. Further, an aqueous solution containing a fatty acid and / or a fatty acid salt may be prepared separately from the reagent and added to the stabilizing reagent before the measurement, or may be added to the latex reagent.
The immunoassay method using magnetic particles for B / F separation is often composed of 2 to 3 liquid systems, but the fatty acid and / or fatty acid of the present invention can be used in any of the constituent reagents as described above. A salt may be contained or added.
本発明で用いる脂肪酸及び／又は脂肪酸塩は、好ましくは、安定化試薬に添加されるのがよい。試料中の非特異吸着物質を吸収する為、特に、本発明で用いる脂肪酸及び／又は脂肪酸塩を溶解した水溶液（すなわち安定化試薬）と試料とを混和し検体を前処理することで非特異反応を回避することができる。 The fatty acid and / or fatty acid salt used in the present invention is preferably added to the stabilizing reagent. In order to absorb non-specifically adsorbed substances in the sample, in particular, a non-specific reaction is performed by pre-treating the sample by mixing the sample with an aqueous solution (ie, stabilizing reagent) in which the fatty acid and / or fatty acid salt used in the present invention is dissolved. Can be avoided.
本発明で使用可能な試料としては特に制限はなく、例えば、全血、血漿、血清、リンパ液、髄液、唾液、汗、涙、腹水、羊水、精液等の体液等も用いることができる。 There is no restriction | limiting in particular as a sample which can be used by this invention, For example, bodily fluids, such as whole blood, plasma, serum, lymph, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, semen, etc. can be used.
本発明の試薬及び方法を用いて測定可能な測定対象物質としては、生体試料中に含まれる生理活性物質であり、抗原抗体反応を利用して測定し得る物質であれば特に限定されず、例えば、タンパク質、糖タンパク質、脂質タンパク質、レセプター、酵素、ウイルス抗原・抗体等が挙げられ、具体的には、ＣＲＰ、ヒトフィブリノーゲン、Ｄダイマー、ＦＤＰ、リウマチ因子、アルファーフェトプロティン（ＡＦＰ）、ＣＥＡ、ＨＢｓ抗原、フェリチン、抗ＨＣＶ抗体、坑ＨＩＶ抗体、抗ストレプトリジンＯ抗体、梅毒トレポネーマ抗体、梅毒脂質抗原に対する抗体、抗ＨＢｓ抗体、抗ＨＢｃ抗体、抗ＨＢｅ抗体等が挙げられる。本発明の用途として好ましくは、ヒト免疫グロブリンを測定対象物質とするキットに適用するのが好ましい。例えば、上述の抗ＨＣＶ抗体、抗ＨＩＶ抗体、抗ストレプトリジンＯ抗体、抗ＨＢｓ抗体、抗ＨＢｃ抗体、抗ＨＢｅ抗体、抗ＴＳＨ抗体、抗Ｔ３抗体、抗Ｔ４抗体等での適用が好ましい。 The measurement target substance that can be measured using the reagent and method of the present invention is not particularly limited as long as it is a physiologically active substance contained in a biological sample and can be measured using an antigen-antibody reaction. , Proteins, glycoproteins, lipid proteins, receptors, enzymes, viral antigens / antibodies, etc., specifically, CRP, human fibrinogen, D-dimer, FDP, rheumatoid factor, alpha-fetoprotein (AFP), CEA, HBs Examples include antigen, ferritin, anti-HCV antibody, anti-HIV antibody, anti-streptridine O antibody, syphilis treponema antibody, antibody against syphilis lipid antigen, anti-HBs antibody, anti-HBc antibody, anti-HBe antibody and the like. The use of the present invention is preferably applied to a kit using human immunoglobulin as a substance to be measured. For example, application with the above-mentioned anti-HCV antibody, anti-HIV antibody, anti-streptolysin O antibody, anti-HBs antibody, anti-HBc antibody, anti-HBe antibody, anti-TSH antibody, anti-T3 antibody, anti-T4 antibody and the like is preferable.
本発明に係る不溶性担体としては、通常この分野で用いられているものであれば特に限定はされないが、例えば、スチレン、塩化ビニル、アクリロニトリル、酢酸ビニル、アクリル酸エステル、メタクリル酸エステル等のビニル系モノマーを重合させてなる単一重合体（例えば、ポリスチレン）の不溶性担体を挙げられる。また、共重合体（例えば、スチレン−スチレンスルホン酸塩共重合体、メタクリル酸重合体、アクリル酸重合体、塩化ビニル−アクリル酸エステル共重合体等）からなる不溶性担体、スチレン−ブタジエン共重合体、メチルメタクリレート−ブタジエン共重合体、アクリロニトリル−ブタジエン共重合体等のブタジエン系共重合体からなる不溶性担体を挙げられる。官能基としてカルボキシル基、１級アミノ基、カルバモイル基（−ＣＯＮＨ２）、水酸基、アルデヒド基等を有し、かつ、基体が上記有機系微粒子からなる不溶性担体を挙げられる。 The insoluble carrier according to the present invention is not particularly limited as long as it is usually used in this field. For example, vinyl type such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylate ester, methacrylate ester, etc. Examples thereof include an insoluble carrier of a single polymer (for example, polystyrene) obtained by polymerizing monomers. Further, an insoluble carrier made of a copolymer (for example, a styrene-styrene sulfonate copolymer, a methacrylic acid polymer, an acrylic acid polymer, a vinyl chloride-acrylic acid ester copolymer, etc.), a styrene-butadiene copolymer And an insoluble carrier made of a butadiene-based copolymer such as methyl methacrylate-butadiene copolymer and acrylonitrile-butadiene copolymer. Examples thereof include an insoluble carrier having a carboxyl group, a primary amino group, a carbamoyl group (—CONH 2 ), a hydroxyl group, an aldehyde group, etc. as a functional group, and a substrate composed of the organic fine particles.
本発明では、不溶性担体として磁性粒子を用いることができる（特開平７−１５１７５５号公報参照）。磁性粒子は、磁気誘導により容易に磁化され得るもので、例えば、四三酸化鉄（Ｆｅ３Ｏ４）、三二酸化鉄（γ−Ｆｅ２Ｏ３）、各種フェライト、鉄、マンガン、ニッケル、コバルト、クロムなどの金属や、コバルト、ニッケル、マンガンなどの合金を内部に含んだポリスチレン、ポリアクリロニトリル、ポリメタクリロニトリル、ポリメタクリル酸メチル、ポリカプラミド、ポリエチレンテレフタレートなどの疎水性重合体、ポリアクリルアミド、ポリメタクリルアミド、ポリビニルピロリドン、ポリビニルアルコール、ポリ（２−オキシエチルアクリレート）、ポリ（２−オキシエチルメタクリレート）、ポリ（２，３−ジオキシプロピルアクリレート）、ポリ（２，３−ジオキシプロピルメタクリレート）、ポリエチレングリコールメタクリレートなどの架橋した親水性重合体、もしくはそれぞれのモノマーの２〜４種程度の共重合体などのラテックス、ゼラチン、リポソームからなる磁性粒子を例示できる。 In the present invention, magnetic particles can be used as the insoluble carrier (see JP-A-7-151755). The magnetic particles can be easily magnetized by magnetic induction. For example, iron trioxide (Fe 3 O 4 ), iron sesquioxide (γ-Fe 2 O 3 ), various ferrites, iron, manganese, nickel, cobalt Hydrophobic polymers such as polystyrene, polyacrylonitrile, polymethacrylonitrile, polymethyl methacrylate, polycapramide, polyethylene terephthalate, polyacrylamide, poly Methacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, poly (2-oxyethyl acrylate), poly (2-oxyethyl methacrylate), poly (2,3-dioxypropyl acrylate), poly (2,3-dioxypropyl methacrylate) , Polyethylene glycol Examples thereof include magnetic particles made of latex, gelatin, and liposomes such as a crosslinked hydrophilic polymer such as dimethacrylate, or about 2 to 4 types of copolymers of each monomer.
不溶性担体のブロッキング処理に使用する成分としては、公知のブロッキング剤を適宜選択して使用できるが、例えば、ウシ血清アルブミン（ＢＳＡ）、ヒト血清アルブミン、カゼイン、スキムミルク、セリシン等のタンパク質成分のブロッキング剤の他、親水性ブロックと疎水性ブロックとからなる合成ポリマーのブロッキング剤を挙げることができる。 As a component used for the blocking treatment of the insoluble carrier, a known blocking agent can be appropriately selected and used. For example, blocking agents for protein components such as bovine serum albumin (BSA), human serum albumin, casein, skim milk, sericin In addition, the blocking agent of the synthetic polymer which consists of a hydrophilic block and a hydrophobic block can be mentioned.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
〔試料〕
以下の実験例では、ヒト血清（試料Ａ−Ｊ、試料群（４０検体））を試料とした。いずれの試料も、別法のイムノブロット法（ＲＩＢＡ III：オーソ社製）により、ＨＣＶ抗体が１Ｃ.Ｏ.Ｉ以下と判定された試料である。「Ｃ.Ｏ.Ｉ」は、ある試料の測定値をカットオフ値で割った値であり、１．０以上を陽性、１．０未満を陰性と判断する、抗体測定における指標である。
〔sample〕
In the following experimental examples, human serum (sample AJ, sample group (40 specimens)) was used as a sample. Each sample is a sample in which the HCV antibody was determined to be 1 C.O.I or less by another immunoblotting method (RIBA III: manufactured by Ortho). “C.O.I” is a value obtained by dividing a measured value of a sample by a cutoff value, and is an index in antibody measurement that judges 1.0 or more as positive and less than 1.0 as negative.
《実験例１》
＜目的＞
本実験例では、脂肪酸含有組成物を使用することで、非特異反応を抑制できるか検討した。更に、脂肪酸含有組成物を使用するのであれば、ブロッキング処理をしないラテックス粒子を使用する場合であっても、非特異反応を抑制できるか検討した。
Example 1
<Purpose>
In this experimental example, it was examined whether a non-specific reaction could be suppressed by using a fatty acid-containing composition. Furthermore, if a fatty acid-containing composition is used, it was investigated whether non-specific reactions can be suppressed even when latex particles that are not subjected to blocking treatment are used.
＜方法＞
安定化試薬とラテックス試薬とからなる２試薬系のラテックス凝集免疫測定法において、脂肪酸としてドデカン酸の非特異抑制効果の検証をした。ドデカン酸は、安定化試薬に含有させた。また、ブロッキング剤であるＢＳＡによるラテックス粒子のブロッキング処理の有無による影響についても確認した。測定は日本電子ＢＭ Ｌ８００自動分析装置を用いた。分析方式に「２ＰＡ」を選択し、試料／安定化試薬／ラテックス試薬の液量はそれぞれ８μＬ／５０μＬ／２４μＬとし、測光ポイントは、４０−６５ポイントを用いた。測定主波長は８４５ｎｍ、計算法方式にはＳＴＤを選択した。具体的な測定工程としては、試料へ安定化試薬を添加した後、５分間反応し、その後、ラテックス試薬を添加・攪拌し５分間反応させ、安定化試薬を添加した後から継続的に吸光度を測定した。結果は表１に示す。
<Method>
In a two-reagent latex agglutination immunoassay method comprising a stabilizing reagent and a latex reagent, the nonspecific suppression effect of dodecanoic acid as a fatty acid was verified. Dodecanoic acid was included in the stabilizing reagent. Moreover, it confirmed also about the influence by the presence or absence of the blocking process of the latex particle by BSA which is a blocking agent. The measurement used JEOL BM L800 automatic analyzer. “2PA” was selected as the analysis method, the sample / stabilizing reagent / latex reagent volume was 8 μL / 50 μL / 24 μL, respectively, and the photometric point was 40-65 points. The measurement dominant wavelength was 845 nm, and STD was selected as the calculation method. Specifically, the stabilization reagent is added to the sample and reacted for 5 minutes, and then the latex reagent is added and stirred to react for 5 minutes. After the stabilization reagent is added, the absorbance is continuously measured. It was measured. The results are shown in Table 1.
＜材料＞
〔安定化試薬の調製〕
脂肪酸を添加した安定化試薬は、２１０ｍｍｏｌ／Ｌ ＮａＣｌ、１．６％ポリオキシエチレンソルビタンモノラウレート（Ｔｗｅｅｎ２０）、２ｍｍｏｌ／Ｌアジ化ナトリウム、２ｍｍｏｌ／Ｌ ＥＤＴＡを含有する４０ｍｍｏｌ／Ｌトリス緩衝液（ｐＨ８．２）に、０．０３％になるようにドデカン酸を溶解して調製した。ドデカン酸は、エタノールを溶媒として１０重量％に調製してから、安定化試薬へ添加した。ドデカン酸有無による効果を検証するため、比較例としてドデカン酸未添加の安定化試薬を調製し測定に使用した。
<Material>
(Preparation of stabilizing reagent)
The stabilizing reagent to which the fatty acid was added was 210 mmol / L NaCl, 1.6% polyoxyethylene sorbitan monolaurate (Tween 20), 2 mmol / L sodium azide, 40 mmol / L Tris buffer solution containing 2 mmol / L EDTA ( It was prepared by dissolving dodecanoic acid at pH 8.2) to 0.03%. Dodecanoic acid was adjusted to 10% by weight using ethanol as a solvent and then added to the stabilizing reagent. In order to verify the effect of the presence or absence of dodecanoic acid, a stabilizing reagent to which no dodecanoic acid was added was prepared and used for measurement as a comparative example.
〔ブロッキング処理なしＨＣＶ抗原固相ラテックス粒子の調製〕
平均粒径０．４８μｍのポリスチレンラテックス粒子（ＪＳＲ社製）を２ｍｍｏｌ／Ｌ ＥＤＴＡ含有の１００ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ８．０）にて１重量％に懸濁した。ポリスチレンラテックス１ｍｇあたり、リコンビナントＨＣＶ抗原（カイロン社製）５μｇを添加し、２５℃３０分間攪拌した。反応液を１４０００ｒｐｍで遠心分離し、ラテックス粒子を１０ｍｍｏｌ／ＬのＭＥＳ緩衝液（ｐＨ６．０）に分散させた。分散は、ソニケーションにより実施した。２５℃３０分間攪拌後、再度遠心分離によりラテックス粒子を回収し、１１７ｍｍｏｌ／Ｌスクロース、２ｍｍｏｌ／Ｌ ＥＤＴＡを含有する１０ｍｍｏｌ／ＬのＭＥＳ緩衝液（ｐＨ６．０）に分散させ、測定に使用した。
[Preparation of HCV antigen solid phase latex particles without blocking treatment]
Polystyrene latex particles (manufactured by JSR) having an average particle size of 0.48 μm were suspended at 1% by weight in 100 mmol / L phosphate buffer (pH 8.0) containing 2 mmol / L EDTA. 5 mg of recombinant HCV antigen (manufactured by Chiron) was added per 1 mg of polystyrene latex, and the mixture was stirred at 25 ° C for 30 minutes. The reaction solution was centrifuged at 14,000 rpm, and the latex particles were dispersed in 10 mmol / L MES buffer (pH 6.0). Dispersion was performed by sonication. After stirring at 25 ° C. for 30 minutes, latex particles were collected again by centrifugation, dispersed in 10 mmol / L MES buffer (pH 6.0) containing 117 mmol / L sucrose and 2 mmol / L EDTA, and used for measurement.
〔ブロッキング処理ありＨＣＶ抗原固相のラテックス粒子の調製〕
平均粒径０．４８μｍのポリスチレンラテックス粒子（ＪＳＲ社製）を２ｍｍｏｌ／Ｌ ＥＤＴＡ含有の１００ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ８．０）にて１重量％に懸濁した。ポリスチレンラテックス１ｍｇあたり、リコンビナントＨＣＶ抗原（カイロン社製）を５μｇ添加し、２５℃３０分間攪拌した。反応液を１４０００ｒｐｍで遠心分離し、ラテックス粒子を１００ｍｍｏｌ／Ｌ Ｔｒｉｓ緩衝液（ｐＨ ８．０）に再懸濁させ、該溶液に０．３重量％になるようにＢＳＡを添加し２５℃３０分間攪拌した。その後遠心分離し、ラテックス粒子を１０ｍｍｏｌ／ＬのＭＥＳ緩衝液（ｐＨ６．０）で再懸濁した。２５℃３０分間攪拌後、遠心し、１１７ｍｍｏｌ／Ｌスクロース、２ｍｍｏｌ／Ｌ ＥＤＴＡを含有する１０ｍｍｏｌ／ＬのＭＥＳ緩衝液（ｐＨ６．０）に再懸濁し測定に使用した。
[Preparation of latex particles of HCV antigen solid phase with blocking treatment]
Polystyrene latex particles (manufactured by JSR) having an average particle size of 0.48 μm were suspended at 1% by weight in 100 mmol / L phosphate buffer (pH 8.0) containing 2 mmol / L EDTA. 5 mg of recombinant HCV antigen (manufactured by Chiron) was added per 1 mg of polystyrene latex, and the mixture was stirred at 25 ° C for 30 minutes. The reaction solution is centrifuged at 14000 rpm, the latex particles are resuspended in 100 mmol / L Tris buffer (pH 8.0), BSA is added to the solution so that the concentration becomes 0.3 wt%, and 25 ° C. for 30 minutes. Stir. Thereafter, the mixture was centrifuged, and the latex particles were resuspended in 10 mmol / L MES buffer (pH 6.0). After stirring at 25 ° C. for 30 minutes, the mixture was centrifuged, resuspended in a 10 mmol / L MES buffer (pH 6.0) containing 117 mmol / L sucrose and 2 mmol / L EDTA, and used for measurement.
＜結果＞
表１に示すとおり、ドデカン酸を使用することにより、（ｉ）非特異反応が強く抑制され（実施例１及び実施例２）、更に、（ii）ラテックス粒子をブロッキングしない場合であっても非特異反応の抑制効果が高いことがわかった（実施例１）。
<Result>
As shown in Table 1, by using dodecanoic acid, (i) non-specific reaction is strongly suppressed (Examples 1 and 2), and (ii) even when latex particles are not blocked, It was found that the effect of suppressing the specific reaction was high (Example 1).
《実験例２》
＜目的＞
本実験例では、ラテックス凝集免疫測定法において、非特異抑制効果とドデカン酸濃度との関係を検討した。
<< Experiment 2 >>
<Purpose>
In this experimental example, in the latex agglutination immunoassay, the relationship between the non-specific inhibitory effect and the dodecanoic acid concentration was examined.
＜方法＞
安定化試薬へのドデカン酸の添加濃度を０．００５〜０．２重量％の範囲で表２のとおり設定した以外は、実験例１と同様に行い、各溶解濃度での非特異抑制効果を検証した。なお、ラテックス粒子は、ブロッキング処理なしで調製したものを使用した。結果は表２に示す。
<Method>
Except that the addition concentration of dodecanoic acid to the stabilizing reagent was set as shown in Table 2 in the range of 0.005 to 0.2% by weight, it was carried out in the same manner as in Experimental Example 1, and the nonspecific inhibition effect at each dissolution concentration Verified. In addition, the latex particle used what was prepared without the blocking process. The results are shown in Table 2.
＜材料＞
実験例１と同様の材料を使用した。
<Material>
The same material as in Experimental Example 1 was used.
＜結果＞
表２に示すとおり、安定化試薬（試料と安定化試薬の液量比＝８μＬ／５０μＬ＝０．１６）におけるドデカン酸の溶解濃度が高いほど、すなわち、０．０２重量％以上、好ましくは、０．０２重量％以上添加されることにより、非特異反応が抑制された。
<Result>
As shown in Table 2, the higher the dissolved concentration of dodecanoic acid in the stabilizing reagent (volume ratio of sample to stabilizing reagent = 8 μL / 50 μL = 0.16), that is, 0.02% by weight or more, preferably By adding 0.02% by weight or more, non-specific reaction was suppressed.
《実験例３》
＜目的＞
本実験例では、ドデカン酸含有組成物の非特異抑制効果を多くの試料で確認した。
<< Experimental Example 3 >>
<Purpose>
In this experimental example, the nonspecific suppression effect of the dodecanoic acid-containing composition was confirmed in many samples.
＜方法＞
別法のイムノブロット法（ＲＩＢＡ III：オーソ社製）で１Ｃ.Ｏ.Ｉ以下とされた試料群（４０検体）について本発明の試薬で測定し、測定値（Ｃ.Ｏ.Ｉ表記）のヒストグラムを得た。
実施例３として、実施例１と同様に調製し、４０検体の測定を実施した。なお、ラテックス粒子は、ブロッキング処理なしで調製したものを使用した。その結果を図１に示す。
比較例３としては、実施例３の試料に対し、比較例１と同様に、脂肪酸を添加しない安定化試薬、及び、ブロッキング処理なしのラテックス粒子を使用した。その結果を図２に示す。
<Method>
A sample group (40 specimens) that was made 1C.O.I or lower by another immunoblotting method (RIBA III: manufactured by Ortho) was measured with the reagent of the present invention, and the measured value (C.O.I notation) A histogram was obtained.
Example 3 was prepared in the same manner as in Example 1, and 40 samples were measured. In addition, the latex particle used what was prepared without the blocking process. The result is shown in FIG.
As Comparative Example 3, as in Comparative Example 1, a stabilizing reagent to which no fatty acid was added and latex particles without blocking treatment were used for the sample of Example 3. The result is shown in FIG.
＜材料＞
実験例１と同様の材料を使用した。
<Material>
The same material as in Experimental Example 1 was used.
＜結果＞
図１及び図２において、横軸はＣ.Ｏ.Ｉ、縦軸は頻度（試料数）を示す。実施例３では比較例３に比べ、陰性検体群の測定値が０Ｃ.Ｏ.Ｉに収束した。非特異反応により測定値が干渉されると、比較例３のようにヒストグラムの測定値の高くなり、０Ｃ.Ｏ.Ｉへの収束が不良となるが、非特異反応が抑制されると実施例３のように測定値が０Ｃ.Ｏ.Ｉへ収束する。
<Result>
1 and 2, the horizontal axis indicates C.O.I, and the vertical axis indicates frequency (number of samples). In Example 3, compared with Comparative Example 3, the measured value of the negative sample group converged to 0 C.O.I. If the measured value is interfered by non-specific reaction, the measured value of the histogram becomes high as in Comparative Example 3, and the convergence to 0C.O.I becomes poor, but when non-specific reaction is suppressed, the example As shown in FIG. 3, the measured value converges to 0C.O.I.
《実験例４》
＜目的＞
本実験例では、ラテックス凝集免疫測定法において、脂肪酸塩含有組成物を使用することで、非特異反応を抑制できるか検討した。
<< Experimental Example 4 >>
<Purpose>
In this experimental example, in the latex agglutination immunoassay, it was examined whether a nonspecific reaction could be suppressed by using a fatty acid salt-containing composition.
＜方法＞
安定化試薬へのオクタン酸ナトリウムの添加濃度を０．０１０〜１．１００重量％の範囲で表３のとおり設定した以外は、実験例１と同様に行い、各溶解濃度での非特異抑制効果を検証した。なお、ラテックス粒子は、ブロッキング処理なしで調製したものを使用した。結果は表３に示す。
<Method>
The nonspecific inhibitory effect at each dissolution concentration was carried out in the same manner as in Experimental Example 1 except that the concentration of sodium octoate added to the stabilizing reagent was set as shown in Table 3 in the range of 0.010 to 1.100% by weight. Verified. In addition, the latex particle used what was prepared without the blocking process. The results are shown in Table 3.
＜材料＞
実験例１と同様の材料を使用した。
<Material>
The same material as in Experimental Example 1 was used.
＜結果＞
表３に示すとおり、脂肪酸塩であるオクタン酸ナトリウムでも脂肪酸と同様に、ブロッキング処理なしでも、強い抑制効果があることがわかった。また、安定化試薬におけるオクタン酸ナトリウムの溶解濃度が高いほど、非特異反応が抑制された。
<Result>
As shown in Table 3, it was found that sodium octoate, which is a fatty acid salt, has a strong inhibitory effect even without a blocking treatment, similar to fatty acids. Moreover, nonspecific reaction was suppressed, so that the dissolution concentration of sodium octanoate in the stabilizing reagent was high.
《実験例５》
＜目的＞
オクタン酸ナトリウム、ノナン酸ナトリウム、ドデカン酸ナトリウム、テトラデカン酸ナトリウム、オクタデカン酸を含有する組成物の非特異抑制効果を検証した。
<< Experimental Example 5 >>
<Purpose>
The nonspecific inhibitory effect of the composition containing sodium octanoate, sodium nonanoate, sodium dodecanoate, sodium tetradecanoate, and octadecanoic acid was verified.
＜方法＞
安定化試薬に表４に示す各濃度の各種脂肪酸又は脂肪酸塩を溶解した以外は、実験例１と同様に行い、各種脂肪酸又は脂肪酸塩の各溶解濃度での非特異抑制効果を検証した。なお、ラテックス粒子は、ブロッキング処理なしで調製したものを使用した。結果は表４に示す。
<Method>
Except that various fatty acids or fatty acid salts of each concentration shown in Table 4 were dissolved in the stabilizing reagent, it was carried out in the same manner as in Experimental Example 1, and the nonspecific inhibitory effect at each dissolved concentration of various fatty acids or fatty acid salts was verified. In addition, the latex particle used what was prepared without the blocking process. The results are shown in Table 4.
＜材料＞
実験例１と同様の材料を使用した。
＜結果＞
表４に示すとおり、（ｉ）オクタン酸ナトリウム、ノナン酸ナトリウム、ドデカン酸ナトリウム、テトラデカン酸ナトリウム、及びオクタデカン酸のいずれを含む組成物においても、ブロッキング処理なしでも、強い抑制効果があることがわかった。また、安定化試薬における前記脂肪酸及び脂肪酸塩の溶解濃度が高いほど、非特異反応が抑制された。また、（ii）脂肪酸及び脂肪酸塩の種類毎で非特異抑制効果を示す至適濃度が異なった。安定化試薬（試料と安定化試薬の液量比＝８μＬ／５０μＬ＝０．１６）への溶解濃度として、オクタン酸ナトリウムは、０．５重量％以上、ノナン酸ナトリウムは、０．５重量％以上、ドデカン酸ナトリウムは、０．１重量％以上、テトラデカン酸ナトリウムは、０．０２重量％以上、オクタデカン酸は、０．０５重量％以上で効果が高かった。至適濃度は、炭素数が少ない脂肪酸及び脂肪酸塩ほど高濃度である傾向があった。
<Material>
The same material as in Experimental Example 1 was used.
<Result>
As shown in Table 4, it was found that (i) a composition containing any of sodium octoate, sodium nonanoate, sodium dodecanoate, sodium tetradecanoate, and octadecanoic acid has a strong inhibitory effect even without blocking treatment. It was. Moreover, nonspecific reaction was suppressed, so that the melt | dissolution concentration of the said fatty acid and fatty acid salt in a stabilization reagent was high. Moreover, the optimal density | concentration which shows a nonspecific inhibitory effect differed for every kind of (ii) fatty acid and fatty acid salt. The dissolution concentration in the stabilizing reagent (volume ratio of the sample and the stabilizing reagent = 8 μL / 50 μL = 0.16) is 0.5% by weight or more for sodium octoate and 0.5% by weight for sodium nonanoate. As described above, sodium dodecanoate was highly effective at 0.1% by weight or more, sodium tetradecanoate was 0.02% by weight or more, and octadecanoic acid was 0.05% by weight or more. The optimum concentration tended to be higher for fatty acids and fatty acid salts with fewer carbon atoms.
以上より、脂肪酸あるいは脂肪酸塩含有組成物を生体由来試料と混合することで非特異反応が抑制されるメカニズムは定かではないが、試料中の非特異因子が脂肪酸により吸収され、非特異因子が不溶性担体へ結合するのを阻害するのではないかと思料している。非特異反応の強弱は、試料供与者毎で異なるが、非特異反応が強い試料程、それを抑制するのに高濃度の脂肪酸の添加が必要となるためである。なお、試料中にも脂肪酸は含まれるが非特異抑制効果を奏すためには十分量ではなく、免疫学的測定試薬に脂肪酸及び／又は脂肪酸塩を添加することで、非特異反応抑制効果を奏すものと思料している。 From the above, the mechanism by which non-specific reactions are suppressed by mixing a fatty acid or fatty acid salt-containing composition with a biological sample is not clear, but non-specific factors in the sample are absorbed by fatty acids and non-specific factors are insoluble. I think that it may inhibit the binding to the carrier. The intensity of the non-specific reaction varies depending on the sample donor, but a sample with a stronger non-specific reaction requires the addition of a higher concentration of fatty acid to suppress it. In addition, although a fatty acid is contained in the sample, it is not a sufficient amount for producing a nonspecific inhibitory effect, and a nonspecific reaction inhibitory effect is obtained by adding a fatty acid and / or a fatty acid salt to an immunological measurement reagent. Thinking of things.
本発明によれば、免疫測定を行う場合に生じる不溶性担体に対する非特異反応を抑制する免疫学的測定試薬及び測定方法を提供することができる。これらの試薬及び方法は、高感度かつ高精度かつ簡便性を求められる臨床検査薬に使用することができる。
例えば、特殊な前処理が不要な臨床検査薬であって、複数の製造メーカーから販売されている自動分析装置に共通してアプリケーションさせることができる。すなわち、複数の機種に共通して採用されている基本的な動作、例えば試料や溶液の分注、反応液の攪拌操作、温度維持、光学的測定の動作で測定が可能な試薬剤形が選択できる。
また、不溶性担体をブロッキング処理することなく、非特異反応を抑制できることから、試薬製造工程が簡略化され、原材料費が低廉化する。また、試料を特殊な前処理をする必要もなく、臨床検査業務の効率化に供する。
ADVANTAGE OF THE INVENTION According to this invention, the immunoassay reagent and the measuring method which suppress the nonspecific reaction with respect to the insoluble support | carrier produced when performing an immunoassay can be provided. These reagents and methods can be used for clinical tests requiring high sensitivity, high accuracy, and simplicity.
For example, it is a clinical test drug that does not require special pretreatment, and can be commonly applied to automatic analyzers sold by a plurality of manufacturers. In other words, select a reagent dosage form that can be measured by basic operations commonly used by multiple models, such as sample and solution dispensing, reaction mixture stirring, temperature maintenance, and optical measurement operations. it can.
In addition, since non-specific reactions can be suppressed without blocking the insoluble carrier, the reagent production process is simplified and the raw material costs are reduced. In addition, there is no need for special pretreatment of the sample, and this contributes to the efficiency of clinical laboratory work.
Claims (8)
次いで、測定対象物質に対する抗原又は抗体を担持させた不溶性担体を含む反応液と反応させる第二の工程とを含み、
脂肪酸及び／又は脂肪酸塩が、安定化試薬及び／又は反応液に溶解されている、請求項７に記載の方法。 A first step of mixing the sample with the stabilizing reagent;
Next, a second step of reacting with a reaction solution containing an insoluble carrier carrying an antigen or antibody against the substance to be measured,
The method according to claim 7, wherein the fatty acid and / or fatty acid salt is dissolved in the stabilizing reagent and / or the reaction solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2012009761A JP5676498B2 (en) | 2012-01-20 | 2012-01-20 | Immunological measuring reagent and measuring method for suppressing non-specific reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2012009761A JP5676498B2 (en) | 2012-01-20 | 2012-01-20 | Immunological measuring reagent and measuring method for suppressing non-specific reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2013148496A JP2013148496A (en) | 2013-08-01 |
| JP5676498B2 true JP5676498B2 (en) | 2015-02-25 |
Family
ID=49046117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2012009761A Active JP5676498B2 (en) | 2012-01-20 | 2012-01-20 | Immunological measuring reagent and measuring method for suppressing non-specific reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5676498B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5541747B2 (en) * | 2012-03-26 | 2014-07-09 | 株式会社Ｌｓｉメディエンス | Immunoassay and kit for small molecules |
| WO2019026569A1 (en) * | 2017-08-01 | 2019-02-07 | 藤倉化成株式会社 | Degradation preventing means for immunoassay reagent containing insoluble carrier particles |
| JP6919499B2 (en) | 2017-08-01 | 2021-08-18 | 藤倉化成株式会社 | Means for Preventing Deterioration of Immunoassay Reagents Containing Insoluble Carrier Particles |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6316266A (en) * | 1986-07-09 | 1988-01-23 | Toyobo Co Ltd | Reagent composition for measurement of autoantibody |
| JPH08211056A (en) * | 1995-02-07 | 1996-08-20 | Konica Corp | Immunity reaction medium composition, enzyme label antibody composition, method for stabilizing composition, immunological measurement method using composition, and kit for immunological measurement method |
| JP4273311B2 (en) * | 2002-04-26 | 2009-06-03 | 株式会社シノテスト | Method and reagent for measuring substance to be measured in sample |
| US20050008851A1 (en) * | 2003-02-18 | 2005-01-13 | Fuji Photo Film Co., Ltd. | Biosensor |
| JP5279357B2 (en) * | 2008-06-12 | 2013-09-04 | キヤノン株式会社 | Composite particle, method for producing the same, dispersion, magnetic biosensing device, and magnetic biosensing method |
-
2012
- 2012-01-20 JP JP2012009761A patent/JP5676498B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2013148496A (en) | 2013-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6182520B2 (en) | Magnetic silica particles, method for measuring a substance to be measured using the magnetic silica particles, and reagent for measuring the substance to be measured | |
| CN105300966A (en) | Preserving fluid and preparation method thereof | |
| JPH02501753A (en) | Manufacturing method of magnetically responsive polymer particles and its applications | |
| JP5362797B2 (en) | Aggregation reaction stabilization method | |
| CA2452766C (en) | Carrier particle latex for assay reagent and assay reagent | |
| JP5676498B2 (en) | Immunological measuring reagent and measuring method for suppressing non-specific reaction | |
| JP5170806B2 (en) | Latex particles for measuring reagent, sensitized latex particles, and measuring reagent for immunoturbidimetric method | |
| JP5844195B2 (en) | Magnetic silica particles, immunoassay using the same, and reagent for immunoassay | |
| JP4167491B2 (en) | Whole blood measurement method | |
| JP2008298505A (en) | Detection method of target substance, mixed particles and detection reagent of target substance | |
| JP6900207B2 (en) | A method for producing a probe-binding carrier and a method for detecting or separating a target substance. | |
| JP5786188B2 (en) | Reagent for measuring C-reactive protein in sample, measuring method, and method for expanding measuring range | |
| JP6496980B2 (en) | Immunoassay method | |
| JP2013125005A (en) | Latex agglutination immunoreagent for ck-mb measurement and measuring method | |
| JP2545707B2 (en) | Immunological diagnostic reagent | |
| JP2005069926A (en) | Magnetic particle for immunological inspection | |
| JP2004325414A (en) | Method and kit for measuring immunity | |
| JP6218916B1 (en) | Magnetic particle dispersion | |
| JP2003344410A (en) | Immuno-measurement reagent and immuno-measurement method | |
| JP2020109148A (en) | Metal complex particle and reagent for immunoassay using the same | |
| JPH11344494A (en) | Immunological agglutination reaction reagent and method for suppressing prozone phenomenon by using the same | |
| JP2020052012A (en) | Immunoassay reagent using insoluble carrier | |
| JP7501872B2 (en) | Magnetic particles and test agents | |
| JP7513345B2 (en) | Magnetic particles and test agents | |
| JP2009069070A (en) | Detecting method of target material, and kit for detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20131107 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20140313 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140422 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140623 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20141216 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20141225 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5676498 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |