JP6132592B2 - Saliva pretreatment liquid - Google Patents
Saliva pretreatment liquid Download PDFInfo
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- JP6132592B2 JP6132592B2 JP2013039897A JP2013039897A JP6132592B2 JP 6132592 B2 JP6132592 B2 JP 6132592B2 JP 2013039897 A JP2013039897 A JP 2013039897A JP 2013039897 A JP2013039897 A JP 2013039897A JP 6132592 B2 JP6132592 B2 JP 6132592B2
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- chloride
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- 239000007788 liquid Substances 0.000 title claims description 108
- 210000003296 saliva Anatomy 0.000 title claims description 101
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 42
- 238000003317 immunochromatography Methods 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 27
- 235000019270 ammonium chloride Nutrition 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- 239000002736 nonionic surfactant Substances 0.000 claims description 22
- 241000894006 Bacteria Species 0.000 claims description 21
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 239000002280 amphoteric surfactant Substances 0.000 claims description 8
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 7
- 235000011147 magnesium chloride Nutrition 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 241000605862 Porphyromonas gingivalis Species 0.000 claims description 4
- 239000006172 buffering agent Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 3
- 229940099596 manganese sulfate Drugs 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 claims description 3
- RNCHQLMELYXRBQ-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2,3-dihydroxybutanedioic acid Chemical compound OCC(N)(CO)CO.OC(=O)C(O)C(O)C(O)=O RNCHQLMELYXRBQ-UHFFFAOYSA-N 0.000 claims description 2
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 claims description 2
- 241000605986 Fusobacterium nucleatum Species 0.000 claims description 2
- 241001135221 Prevotella intermedia Species 0.000 claims description 2
- 241000589892 Treponema denticola Species 0.000 claims description 2
- GZLGNNHEHXBCBI-UHFFFAOYSA-L [Na+].[Na+].OC(=O)C(O)C(O)C(O)=O.[O-]C(=O)C(O)C(O)C([O-])=O Chemical compound [Na+].[Na+].OC(=O)C(O)C(O)C(O)=O.[O-]C(=O)C(O)C(O)C([O-])=O GZLGNNHEHXBCBI-UHFFFAOYSA-L 0.000 claims description 2
- HDFXRQJQZBPDLF-UHFFFAOYSA-L disodium hydrogen carbonate Chemical compound [Na+].[Na+].OC([O-])=O.OC([O-])=O HDFXRQJQZBPDLF-UHFFFAOYSA-L 0.000 claims description 2
- CSVGEMRSDNSWRF-UHFFFAOYSA-L disodium;dihydrogen phosphate Chemical compound [Na+].[Na+].OP(O)([O-])=O.OP(O)([O-])=O CSVGEMRSDNSWRF-UHFFFAOYSA-L 0.000 claims description 2
- KVMLCRQYXDYXDX-UHFFFAOYSA-M potassium;chloride;hydrochloride Chemical compound Cl.[Cl-].[K+] KVMLCRQYXDYXDX-UHFFFAOYSA-M 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- 241001470488 Tannerella Species 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 claims 1
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 48
- 238000002156 mixing Methods 0.000 description 45
- 239000012528 membrane Substances 0.000 description 31
- 238000000034 method Methods 0.000 description 31
- 238000012360 testing method Methods 0.000 description 27
- 239000007864 aqueous solution Substances 0.000 description 21
- 239000007853 buffer solution Substances 0.000 description 21
- -1 polyoxyethylene Polymers 0.000 description 18
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 17
- 239000008363 phosphate buffer Substances 0.000 description 17
- PIIRYSWVJSPXMW-UHFFFAOYSA-N 1-octyl-4-(4-octylphenoxy)benzene Chemical compound C1=CC(CCCCCCCC)=CC=C1OC1=CC=C(CCCCCCCC)C=C1 PIIRYSWVJSPXMW-UHFFFAOYSA-N 0.000 description 16
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 16
- 239000002202 Polyethylene glycol Substances 0.000 description 16
- 229920001223 polyethylene glycol Polymers 0.000 description 16
- 239000000427 antigen Substances 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 235000014113 dietary fatty acids Nutrition 0.000 description 12
- 239000000194 fatty acid Substances 0.000 description 12
- 229930195729 fatty acid Natural products 0.000 description 12
- 238000001514 detection method Methods 0.000 description 9
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 102000015728 Mucins Human genes 0.000 description 4
- 108010063954 Mucins Proteins 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000003945 anionic surfactant Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 3
- 229920000053 polysorbate 80 Polymers 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 150000003973 alkyl amines Chemical class 0.000 description 2
- 150000005215 alkyl ethers Chemical class 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 2
- 238000007781 pre-processing Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 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 2
- GUQQBLRVXOUDTN-XOHPMCGNSA-N 3-[dimethyl-[3-[[(4r)-4-[(3r,5s,7r,8r,9s,10s,12s,13r,14s,17r)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CC(O)CS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 GUQQBLRVXOUDTN-XOHPMCGNSA-N 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-N Carbamic acid Chemical class NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 1
- QZSODHGHTMLVCM-UHFFFAOYSA-N Cl.Cl.[K] Chemical compound Cl.Cl.[K] QZSODHGHTMLVCM-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 241000194019 Streptococcus mutans Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- QTSOZIIBRLXIJQ-UHFFFAOYSA-N [Na].[Na].CCCCCCCCCCCCN(CCC(O)=O)CCC(O)=O Chemical compound [Na].[Na].CCCCCCCCCCCCN(CCC(O)=O)CCC(O)=O QTSOZIIBRLXIJQ-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 125000005037 alkyl phenyl group Chemical group 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 208000034391 chronic adult periodontitis Diseases 0.000 description 1
- 208000001277 chronic periodontitis Diseases 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 description 1
- SYELZBGXAIXKHU-UHFFFAOYSA-N dodecyldimethylamine N-oxide Chemical compound CCCCCCCCCCCC[N+](C)(C)[O-] SYELZBGXAIXKHU-UHFFFAOYSA-N 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- MTNDZQHUAFNZQY-UHFFFAOYSA-N imidazoline Chemical compound C1CN=CN1 MTNDZQHUAFNZQY-UHFFFAOYSA-N 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- CGVLVOOFCGWBCS-RGDJUOJXSA-N n-octyl β-d-thioglucopyranoside Chemical compound CCCCCCCCS[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O CGVLVOOFCGWBCS-RGDJUOJXSA-N 0.000 description 1
- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 description 1
- 230000003239 periodontal effect Effects 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 description 1
- FYKDNWHPKQOZOT-UHFFFAOYSA-M sodium;dihydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OP(O)([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FYKDNWHPKQOZOT-UHFFFAOYSA-M 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- FBWNMEQMRUMQSO-UHFFFAOYSA-N tergitol NP-9 Chemical compound CCCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCOCCOCCOCCOCCO)C=C1 FBWNMEQMRUMQSO-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Description
本発明は、免疫クロマトグラフィー法によりヒトまたは動物の唾液中の歯周病原細菌を同定・定量を行うために用いる唾液の前処理液に関する。 The present invention relates to a pretreatment solution for saliva used for identifying and quantifying periodontopathic bacteria in human or animal saliva by immunochromatography.
従来から、細菌の検査には抗原抗体反応を利用した検査が行われてきた。一般的な方法として免疫クロマトグラフィー法がある（例えば、特許文献１〜３参照。）。この方法では、ニトロセルロースなどの多孔質膜（孔径：数十μｍ）の片端に目的とする特定の抗原のみに付く特定の抗体（以後、特異抗体と記す。）が染み込まされており、多孔質膜の中程には同様に特定の抗原のみに付く別の特異抗体が帯状に染み込まされて多孔質膜に固定されている。片端に染み込まされている特異抗体は、予め金コロイド等の粒子で着色されており、その特異抗体が存在している多孔質膜の片端上にサンプル液を染み込ませるとサンプル液中に特異抗体と反応する抗原があれば、その抗原は特異抗体と結び付いて着色粒子を付けた状態で多孔質膜を毛細管現象によってサンプル液を染み込ませた側と反対の片端へ向かって移動して行く。移動の途中で帯状に固定されている別の特異抗体の個所を通過する際に、抗原はその別の特異抗体に捕捉され、多孔質膜上に帯状の染みが現れる。このことによって目的の抗原がサンプル中に存在していること及びその量を知ることができる。 Conventionally, a test using an antigen-antibody reaction has been performed for a bacterial test. As a general method, there is an immunochromatography method (see, for example, Patent Documents 1 to 3). In this method, a specific antibody (hereinafter referred to as a specific antibody) attached to only a specific antigen of interest is impregnated on one end of a porous membrane (pore diameter: several tens of μm) such as nitrocellulose. Similarly, in the middle of the membrane, another specific antibody attached only to a specific antigen is infiltrated in a band shape and fixed to the porous membrane. The specific antibody soaked at one end is colored in advance with particles such as colloidal gold, and when the sample liquid is soaked on one end of the porous membrane where the specific antibody exists, the specific antibody is in the sample liquid. If there is an antigen that reacts, the antigen moves to one end opposite to the side where the sample solution is impregnated by capillary action in a state where the antigen is bound to the specific antibody and colored particles are attached. When passing through the location of another specific antibody fixed in a band shape in the course of movement, the antigen is captured by the other specific antibody, and a band-like stain appears on the porous membrane. This makes it possible to know that the target antigen is present in the sample and its amount.
このような技術を応用すれば前述の口腔内細菌の同定・定量を行うことができそうであるが、口腔内細菌の検査に用いられる主要なサンプルは唾液であるため、唾液中に存在するムチンと呼ばれる高粘性物質及び検出の対象ではない連鎖球菌が多孔質膜の孔を塞いでしまい、またムチンは唾液中に存在する口腔粘膜面から剥がれ落ちた上皮付着細胞を凝集させる働きもするため、これらの物質により多孔質膜の孔が塞がれて口腔内細菌を通過させることができなかった。そのため、本出願人は、以前に唾液の前処理キット及び唾液の前処理方法を提案した（特許文献４参照。）。 If such a technique is applied, it is likely that the above-mentioned oral bacteria can be identified and quantified. However, since the main sample used for the inspection of oral bacteria is saliva, mucins present in saliva Because the high-viscosity substance called and streptococci that are not the target of detection block pores of the porous membrane, and mucin also acts to aggregate epithelial adherent cells that have fallen off the oral mucosal surface present in saliva, These substances blocked the pores of the porous membrane and could not allow oral bacteria to pass through. Therefore, the present applicant has previously proposed a pretreatment kit for saliva and a pretreatment method for saliva (see Patent Document 4).
近年では、特に成人性歯周炎の原因菌としてポルフィロモナス・ジンジバリス（以下、Ｐｇ菌と記すことがある。）が重要視されているため、ヒトまたは動物の唾液中のＰｇ菌の有無や量を簡便に検査できれば罹患リスクや現状の罹患状況の把握ができ、極めて多くの人々に有効な情報を与えることが可能である。しかしながら、前記唾液処理方法は特にミュータンス連鎖球菌に対しては有効なものであるものの、Ｐｇ菌の検出に用いるとＰｇ菌の菌体表面を変性させてしまい、免疫クロマトグラフィー法による検出はできなかった。 In recent years, since Porphyromonas gingivalis (hereinafter sometimes referred to as Pg fungus) is regarded as an important causative agent of adult periodontitis, the presence or absence of Pg fungus in human or animal saliva, If the amount can be easily examined, it is possible to grasp the risk of morbidity and the current morbidity, and it is possible to provide useful information to an extremely large number of people. However, the saliva treatment method is particularly effective against Streptococcus mutans, but when used for detection of Pg bacteria, the cell surface of Pg bacteria is denatured and cannot be detected by immunochromatography. There wasn't.
抗原抗体反応を利用した免疫クロマトグラフィー法を用いてヒトまたは動物の唾液中の歯周病原細菌を同定・定量を行う際の前記欠点を解消し、簡便な方法で唾液中のムチン及び連鎖球菌の凝集を取り除くことが可能で、且つ歯周病原細菌の菌体表面を変性させないような処理が可能な唾液の前処理液を提供することを課題とする。 Eliminates the drawbacks of identifying and quantifying periodontopathic bacteria in human or animal saliva using an immunochromatography method that utilizes an antigen-antibody reaction, and a simple method for the detection of mucin and streptococci in saliva It is an object of the present invention to provide a pretreatment solution for saliva that can remove aggregation and that can be treated so as not to denature the surface of periodontopathic bacteria.
本発明者等は前記課題を解決すべく鋭意検討を重ねた結果、特定の配合の液で前処理を行うことにより、唾液中の歯周病原細菌の菌体表面を変性させずに唾液を処理し、後の免疫クロマトグラフィー法による検出が可能になることを見出して本発明を完成した。 As a result of intensive studies to solve the above-mentioned problems, the present inventors processed saliva without degenerating the cell surface of periodontopathic bacteria in saliva by performing pretreatment with a liquid having a specific composition. The present invention was completed by finding that detection by the subsequent immunochromatography method becomes possible.
本発明に係る唾液の前処理液は、用いることで簡便に唾液中のムチン及び連鎖球菌の凝集による多孔質膜の孔の目詰まりを防止することが可能であり、且つ、歯周病原細菌の菌体表面を変性させることがないため抗体反応を明確に確認することができる優れた唾液の前処理液である。 The saliva pretreatment liquid according to the present invention can be used to easily prevent clogging of pores of the porous membrane due to aggregation of mucin and streptococci in saliva, and It is an excellent pretreatment solution for saliva that can clearly confirm the antibody reaction because it does not denature the cell surface.
本発明に係る唾液の前処理液には、非イオン性界面活性剤及び／または両性界面活性剤が配合される。これは、歯周病原細菌表面のタンパク質を菌体表面を変性させることなく可溶化し、歯周病原細菌が多孔質膜中を効率よく通り抜けることを可能とする。 In the pretreatment liquid for saliva according to the present invention, a nonionic surfactant and / or an amphoteric surfactant is blended. This solubilizes the protein on the surface of periodontopathic bacteria without denaturing the cell surface, allowing periodontopathic bacteria to pass through the porous membrane efficiently.
非イオン性界面活性剤は、ポリエチレングリコールモノオクチルフェニルエーテル、n−オクチル−β−Ｄ−グルコシド、ｎ−へプチル−β−Ｄ−チオグルコシド、ｎ−オクチル−β−Ｄ−チオグルコシド、ノニルフェノキシポリエトキシエタノール、ポリオキシエチレンソルビタンモノオレエート、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンアルキルフェニルエーテル、ポリオキシエチレンステロールエーテル、ポリオキシエチレンポリオキシプロピレンブロックコポリマー、ポリオキシエチレンポリオキシプロピレンアルキルエーテル、ポリオキシエチレングリセリン脂肪酸エステル、ポリオキシエチレンヒマシ油、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ソルビトール脂肪酸エステル、ポリエチレングリコール脂肪酸エステル、エチレングリコール脂肪酸エステル、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル、ソルビタン脂肪酸エステル、プロピレングリコール脂肪酸エステル、ショ糖脂肪酸エステル、脂肪酸アルカノールアミド、ポリオキシエチレン脂肪酸アミド、ポリオキシエチレンアルキルアミン、アルキルフェノール−ホルムアルデヒド縮合物のエチレンオキサイド付加物からなる群より選ばれる１種または２種以上であることが好ましい。 Nonionic surfactants include polyethylene glycol monooctyl phenyl ether, n-octyl-β-D-glucoside, n-heptyl-β-D-thioglucoside, n-octyl-β-D-thioglucoside, nonylphenoxy Polyethoxyethanol, polyoxyethylene sorbitan monooleate, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene sterol ether, polyoxyethylene polyoxypropylene block copolymer, polyoxyethylene polyoxypropylene alkyl ether, poly Oxyethylene glycerin fatty acid ester, polyoxyethylene castor oil, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyester Tylene glycol fatty acid ester, ethylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester, fatty acid alkanolamide, polyoxyethylene fatty acid amide, polyoxyethylene alkylamine, alkylphenol -It is preferable that it is 1 type, or 2 or more types chosen from the group which consists of an ethylene oxide adduct of formaldehyde condensate.
両性界面活性剤は、ＣＨＡＰＳ（３−［(３−コラミドプロピル)−ジメチルアンモニオ］−１−プロパンスルホナート、ＣＨＡＰＳＯ（３−［（３−コラミドプロピル）−ジメチルアンモニオ］−２−ヒドロキシ−１−プロパンスルホナート）、カルボキシベタイン、イミダゾリニウムベタイン、Ｎ−ドデシル−Ｎ−（２−カルボキシエチル）−β―アラニンジナトリウム等のアミノカルボン酸塩、Ｎ,Ｎ−ジメチルドデシルアミンオキシド等のアルキルアミンオキシドからなる群より選ばれる１種または２種以上であることが好ましい。 Amphoteric surfactants include CHAPS (3-[(3-colamidopropyl) -dimethylammonio] -1-propanesulfonate, CHAPSO (3-[(3-colamidopropyl) -dimethylammonio] -2- Hydroxy-1-propanesulfonate), carboxybetaines, imidazolinium betaines, aminocarboxylates such as N-dodecyl-N- (2-carboxyethyl) -β-alanine disodium, N, N-dimethyldodecylamine oxide It is preferable that it is 1 type, or 2 or more types chosen from the group which consists of alkylamine oxides.
従来から免疫クロマトグラフィー法では、サンプル液や抗原液が検査器具内をスムーズに移動できるように陰イオン界面活性剤を使用することがよくある。しかし、本発明に係る歯周病原細菌の同定・定量を行うための唾液の前処理液に使用される界面活性剤は、実験の結果から非イオン性界面活性剤及び／または両性界面活性剤である必要があり、ラウリル硫酸ナトリウムやドデシルベンゼンスルホン酸ナトリウム等の陰イオン界面活性剤では、抗体による抗原検出を行うことができない。 Conventionally, in an immunochromatography method, an anionic surfactant is often used so that a sample solution or an antigen solution can move smoothly in a test instrument. However, the surfactant used in the saliva pretreatment liquid for identifying and quantifying the periodontal pathogenic bacteria according to the present invention is a nonionic surfactant and / or an amphoteric surfactant based on the experimental results. It is necessary that an anionic surfactant such as sodium lauryl sulfate or sodium dodecylbenzenesulfonate cannot detect an antigen using an antibody.
本発明に係る唾液の前処理液において、非イオン性界面活性剤及び／または両性界面活性剤は、唾液の前処理液に対して０．０２〜２５重量％の配合量になるように配合されることが好ましく、０．０２重量％未満では抗原抗体反応による検出感度が低くなる傾向があり、２５重量％を超えても抗原抗体反応による検出感度が低下し易いため好ましくない。 In the saliva pretreatment liquid according to the present invention, the nonionic surfactant and / or the amphoteric surfactant are blended so as to have a blending amount of 0.02 to 25% by weight with respect to the saliva pretreatment liquid. If the amount is less than 0.02% by weight, the detection sensitivity due to the antigen-antibody reaction tends to be low, and if it exceeds 25% by weight, the detection sensitivity due to the antigen-antibody reaction tends to decrease.
本発明に係る唾液の前処理液に配合される、塩化ナトリウム、塩化カリウム、塩化カル
シウム、塩化マグネシウム、硫酸マグネシウム、硫酸マンガン、塩化アンモニウムからな
る群より選ばれる１種または２種以上の物質は、抗原ではない異物が抗体と反応して検出
されてしまう偽陽性を抑制する効果があり、免疫クロマトグラフィー法による検出をより
正確にする効果がある。その濃度は唾液の前処理液中に０．２５〜５ｍｏｌ／Ｌである。
０．２５ｍｏｌ／Ｌ未満では偽陽性を抑える効果が少なく、５ｍｏｌ／Ｌを超えると抗原
抗体反応に影響が出て感度が低下してしまうため適さない。
One or more substances selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, magnesium sulfate, manganese sulfate, and ammonium chloride, which are blended in the saliva pretreatment liquid according to the present invention, This has the effect of suppressing false positives in which foreign substances that are not antigens are detected by reacting with antibodies, and has the effect of making detection by immunochromatography more accurate. The concentration is 0.25 to 5 mol / L in the pretreatment solution of saliva.
If it is less than 0.25 mol / L, the effect of suppressing false positives is small, and if it exceeds 5 mol / L, the antigen-antibody reaction is affected and sensitivity is lowered, which is not suitable.
本発明に係る唾液の前処理液は、抗原である歯周病原細菌の多孔質膜中の移動を容易とするために、ｐＨ３〜８であることが必要である。ｐＨが３未満であると偽陽性が強く現れ検出精度が低下してしまい、一方、ｐＨ８を超えると抗原の菌体表面に変性が生じてしまい、抗体による抗原検出ができなくなる。 The saliva pretreatment liquid according to the present invention needs to have a pH of 3 to 8 in order to facilitate movement of periodontopathic bacteria, which are antigens, in the porous membrane. If the pH is less than 3, false positives appear strongly and the detection accuracy is lowered. On the other hand, if the pH is more than 8, denaturation occurs on the surface of the antigen cell, making it impossible to detect the antigen using antibodies.
そのため、本発明に係る唾液の前処理液には、緩衝作用付与物質及び水を配合している。緩衝作用付与物質は、２以上の物質の組み合わせからなり、塩酸−塩化カリウム，塩酸−トリス（ヒドロキシメチル）アミノメタン，塩酸−グリシン，グリシン−水酸化ナトリウム、クエン酸−リン二水素ナトリウム、クエン酸−クエン酸ナトリウム，リン酸二水素ナトリウム−リン酸二水素ナトリウム，炭酸ナトリウム−重炭酸ナトリウム，酢酸−酢酸ナトリウム，酒石酸−酒石酸ナトリウム，酒石酸−トリス（ヒドロキシメチル）アミノメタン等が例示される。この群より選ばれる１種または２種以上が配合されることが好ましい。 Therefore, the pretreatment liquid for saliva according to the present invention contains a buffering substance and water. The buffering substance is a combination of two or more substances: hydrochloric acid-potassium chloride, hydrochloric acid-tris (hydroxymethyl) aminomethane, hydrochloric acid-glycine, glycine-sodium hydroxide, citric acid-sodium dihydrogen phosphate, citric acid -Sodium citrate, sodium dihydrogen phosphate-sodium dihydrogen phosphate, sodium carbonate-sodium bicarbonate, acetic acid-sodium acetate, tartaric acid-sodium tartrate, tartaric acid-tris (hydroxymethyl) aminomethane and the like. It is preferable that 1 type, or 2 or more types selected from this group is blended.
本発明に係る唾液の前処理液中の緩衝作用付与物質の濃度は、単位体積当たりの、緩衝作用付与物質として配合されるすべての物質の物質量の総和として定義され、前記ｐＨの前提から唾液の前処理液中に０．０１〜３ｍｏｌ／Ｌの範囲となることが好ましい。 The concentration of the buffer action-imparting substance in the saliva pretreatment liquid according to the present invention is defined as the sum of the substance amounts of all substances blended as the buffer action-imparting substance per unit volume. It is preferable that it becomes the range of 0.01-3 mol / L in this pre-processing liquid.
本発明に係る唾液の前処理液が対象としている歯周病原細菌は、具体的には、ポルフィロモナス・ジンジバリス、プレボテラ・インターメディア、アグリゲイティバクター・アクチノミセテムコミタンス、フゾバクテリウム・ヌクレアタム、トレポネーマ・デンティコーラ、タンネレラ・フォーサイシアからなる群より選ばれる１種または２種以上である。中でもポルフィロモナス・ジンジバリスに最も効果がある。 Periodontopathic bacteria targeted by the saliva pretreatment liquid according to the present invention are specifically Porphyromonas gingivalis, Prevotella intermedia, Aggregate bacter actinomycetemcomitans, Fusobacterium nucleatum, One or more selected from the group consisting of Treponema Denticola and Tannerrella Forsaicia. Among them, it is most effective for Porphyromonas gingivalis.
以下、本発明に係る唾液の前処理液に関する実施例を示す。なお、本発明は、下記に記された唾液の前処理液に限定されるものではない。 Hereinafter, the Example regarding the pre-processing liquid of the saliva which concerns on this invention is shown. The present invention is not limited to the saliva pretreatment liquid described below.
免疫クロマトグラフィー法による患者の唾液中のＰｇ菌数を測定する試験を行った。具体的には、抗体反応の検出の有無を観察した。なお、同じ患者の唾液中のＰｇ菌数をリアルタイムＰＣＲ法により予め計測し、９．７２×１０５ｃｅｌｌｓ／ｍＬであることを確認した。この値の菌数を検出することができれば、実際の免疫クロマトグラフィー法による検査においても検出することが可能である。 A test for measuring the number of Pg bacteria in the saliva of patients by immunochromatography was conducted. Specifically, the presence or absence of detection of antibody reaction was observed. In addition, the number of Pg bacteria in the saliva of the same patient was measured in advance by a real-time PCR method, and confirmed to be 9.72 × 10 5 cells / mL. If the number of bacteria of this value can be detected, it can also be detected in an actual immunochromatographic test.
免疫クロマトグラフィー法による試験は公知の方法により行った。多孔質膜としてニトロセルロースメンブレン（商品名：ＳＸＨＦ、日本ミリポア社製）を５ｍｍ×２５ｍｍの長方形に切り出したものを使用した。この多孔質膜に、抗Ｐｇ菌抗体と、前記抗Ｐｇ菌抗体とは異なる別の抗Ｐｇ菌抗体を粒径４０ｎｍの金コロイド（British Biocell International社製）で標識化したものを塗布した。唾液２５０μＬを以下に示す方法で作製した唾液の前処理液で処理し試験を行った。
＜実施例１＞
The immunochromatographic test was performed by a known method. As the porous membrane, a nitrocellulose membrane (trade name: SXHF, manufactured by Nihon Millipore) cut into a 5 mm × 25 mm rectangle was used. This porous membrane was coated with an anti-Pg bacterium antibody and another anti-Pg bacterium antibody different from the anti-Pg bacterium antibody labeled with gold colloid (British Biocell International) having a particle size of 40 nm. A test was performed by treating 250 μL of saliva with a pretreatment solution of saliva prepared by the method described below.
<Example 1>
０．１ｍｏｌ／Ｌクエン酸２９．４ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２０．６ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
29.4 mL of 0.1 mol / L citric acid and 20.6 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ４．２の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment solution of pH 4.2 was prepared by mixing equal amounts of A solution and B solution.
唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例２＞
100 μL of saliva pretreatment solution added to 250 μL of saliva and mixed were tested using immunochromatography. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 2>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例３＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 3>
０．１ｍｏｌ／Ｌクエン酸１９．７ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム３０．３ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
19.7 mL of 0.1 mol / L citric acid and 30.3 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５．８の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment liquid of saliva having a pH of 5.8 was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例４＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 4>
０．１ｍｏｌ／Ｌクエン酸６．５ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム４３．６ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
6.5 mL of 0.1 mol / L citric acid and 43.6 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ７の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment liquid of pH 7 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例５＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 5>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化ナトリウム濃度が１ｍｏｌ／Ｌとなるように塩化ナトリウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Sodium chloride was added to this buffer solution so that the sodium chloride concentration would be 1 mol / L to obtain solution A.
両性界面活性剤としてＣＨＡＰＳ（３−［(３−コラミドプロピル)−ジメチルアンモニオ］−１−プロパンスルホナートを水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As an amphoteric surfactant, CHAPS (3-[(3-collamidopropyl) -dimethylammonio] -1-propanesulfonate was added to water to give a 5 wt% aqueous solution, which was designated as solution B.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例６＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 6>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化ナトリウム濃度が２ｍｏｌ／Ｌとなるように塩化ナトリウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Sodium chloride was added to this buffer solution so that the sodium chloride concentration would be 2 mol / L to obtain solution A.
非イオン性界面活性剤としてポリオキシエチレンソルビタンモノオレエートを水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
Liquid B was prepared by adding polyoxyethylene sorbitan monooleate as a nonionic surfactant to water to form a 5% by weight aqueous solution.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例７＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 7>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化ナトリウム濃度が３ｍｏｌ／Ｌとなるように塩化ナトリウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Sodium chloride was added to this buffer solution so that the sodium chloride concentration would be 3 mol / L to obtain solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例８＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 8>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化ナトリウム濃度が４ｍｏｌ／Ｌとなるように塩化ナトリウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Sodium chloride was added to this buffer solution so that the sodium chloride concentration was 4 mol / L to obtain solution A.
両性界面活性剤としてＣＨＡＰＳ（３−［(３−コラミドプロピル)−ジメチルアンモニオ］−１−プロパンスルホナートを水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As an amphoteric surfactant, CHAPS (3-[(3-collamidopropyl) -dimethylammonio] -1-propanesulfonate was added to water to give a 5 wt% aqueous solution, which was designated as solution B.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
体反応は確認された。
＜実施例９＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
Body reaction was confirmed.
<Example 9>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し１０重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As a nonionic surfactant, polyethylene glycol monooctylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was added to water to give a 10% by weight aqueous solution.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１０＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 10>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し２０重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As a nonionic surfactant, polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was added to water to give a 20% by weight aqueous solution, which was designated as solution B.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１１＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 11>
０．１ｍｏｌ／Ｌ酢酸４．８ｍＬと０．１ｍｏｌ／Ｌ酢酸ナトリウム４５．２ｍＬを混合し、酢酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
Acetic acid buffer was prepared by mixing 4.8 mL of 0.1 mol / L acetic acid and 45.2 mL of 0.1 mol / L sodium acetate.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５．６の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment liquid of saliva having a pH of 5.6 was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１２＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 12>
０．１ｍｏｌ／Ｌ酢酸４．８ｍＬと０．１ｍｏｌ／Ｌ酢酸ナトリウム４５．２ｍＬを混合し、酢酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
Acetic acid buffer was prepared by mixing 4.8 mL of 0.1 mol / L acetic acid and 45.2 mL of 0.1 mol / L sodium acetate.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５．６の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment liquid of saliva having a pH of 5.6 was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１３＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 13>
０．２ｍｏｌ／Ｌリン酸二水素ナトリウム９２ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム８ｍＬを合わせ、リン酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
A phosphate buffer was prepared by combining 92 mL of 0.2 mol / L sodium dihydrogen phosphate and 8 mL of 0.2 mol / L disodium hydrogen phosphate.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し１０重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５．８の唾液の前処理液を作製した。
As a nonionic surfactant, polyethylene glycol monooctylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was added to water to give a 10% by weight aqueous solution.
A pretreatment liquid of saliva having a pH of 5.8 was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１４＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 14>
０．１mol／Ｌトリス（ヒドロキシメチル）アミノメタン水溶液５０ｍＬと０．１mol／Ｌ塩酸４４．２ｍＬを合わせ、トリス（ヒドロキシメチル）アミノメタン−塩酸バッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
50 mL of 0.1 mol / L tris (hydroxymethyl) aminomethane aqueous solution and 44.2 mL of 0.1 mol / L hydrochloric acid were combined to prepare a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し１０重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ７．２の唾液の前処理液を作製した。
As a nonionic surfactant, polyethylene glycol monooctylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was added to water to give a 10% by weight aqueous solution.
A pretreatment liquid of pH 7.2 was prepared by mixing A liquid and B liquid in equal amounts.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１５＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 15>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬ合わせ、クエン酸リン酸バッファを作製した。
この緩衝液に塩化カリウム濃度が１ｍｏｌ／Ｌとなるように塩化カリウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were combined to prepare a citrate phosphate buffer.
Potassium chloride was added to this buffer solution so that the potassium chloride concentration was 1 mol / L to prepare solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し１０重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As a nonionic surfactant, polyethylene glycol monooctylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was added to water to give a 10% by weight aqueous solution.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１６＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 16>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化マグネシウム濃度が１ｍｏｌ／Ｌとなるように塩化マグネシウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Magnesium chloride was added to this buffer solution so that the magnesium chloride concentration would be 1 mol / L to obtain Solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し２０重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As a nonionic surfactant, polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was added to water to give a 20% by weight aqueous solution, which was designated as solution B.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１７＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 17>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に硫酸マグネシウム濃度が０．５ｍｏｌ／Ｌとなるように硫酸マグネシウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Magnesium sulfate was added to this buffer solution so that the magnesium sulfate concentration would be 0.5 mol / L to obtain Solution A.
非イオン性界面活性剤としてポリオキシエチレンソルビタンモノオレエートを水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
Liquid B was prepared by adding polyoxyethylene sorbitan monooleate as a nonionic surfactant to water to form a 5% by weight aqueous solution.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜実施例１８＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Example 18>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に硫酸マグネシウム濃度が１ｍｏｌ／Ｌとなるように硫酸マグネシウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Magnesium sulfate was added to this buffer solution so that the magnesium sulfate concentration would be 1 mol / L to obtain Solution A.
両性界面活性剤としてＣＨＡＰＳ（３−［(３−コラミドプロピル)−ジメチルアンモニオ］−１−プロパンスルホナートを水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As an amphoteric surfactant, CHAPS (3-[(3-collamidopropyl) -dimethylammonio] -1-propanesulfonate was added to water to give a 5 wt% aqueous solution, which was designated as solution B.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。多孔質膜の目詰まりはなく、抗体反応は明確に確認できた。
＜比較例１＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. There was no clogging of the porous membrane, and the antibody reaction was clearly confirmed.
<Comparative Example 1>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液に塩化マグネシウム濃度が１ｍｏｌ／Ｌとなるように塩化マグネシウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Magnesium chloride was added to this buffer solution so that the magnesium chloride concentration would be 1 mol / L to obtain Solution A.
陰イオン界面活性剤としてラウリル硫酸ナトリウム５重量％を水に添加しＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As an anionic surfactant, 5% by weight of sodium lauryl sulfate was added to water to prepare a solution B.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。抗体反応は全く確認できなかった。
＜比較例２＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. No antibody reaction could be confirmed.
<Comparative example 2>
０．２ｍｏｌ／Ｌ塩化カリウム５０ｍＬと０．２ｍｏｌ／Ｌ塩酸６．７ｍＬとを混合し、塩酸−塩酸カリウムバッファを作製した。
この緩衝液に塩化アンモニウム濃度が０．５ｍｏｌ／Ｌとなるように塩化アンモニウムを添加しＡ液とした。
A hydrochloric acid-potassium hydrochloride buffer was prepared by mixing 50 mL of 0.2 mol / L potassium chloride and 6.7 mL of 0.2 mol / L hydrochloric acid.
Ammonium chloride was added to this buffer solution so that the ammonium chloride concentration was 0.5 mol / L to prepare a solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し５重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ２．２の唾液の前処理液を作製した。
Liquid B was obtained by adding polyethylene glycol monooctyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) as a nonionic surfactant to water to make a 5 wt% aqueous solution.
A pretreatment liquid of saliva having a pH of 2.2 was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。抗体反応は全く確認できなかった。
＜比較例３＞
A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. No antibody reaction could be confirmed.
<Comparative Example 3>
０．１ｍｏｌ／Ｌクエン酸２４．３ｍＬと０．２ｍｏｌ／Ｌリン酸水素二ナトリウム２５．７ｍＬを混合し、クエン酸リン酸バッファを作製した。
この緩衝液にリン酸カルシウム濃度が０．５ｍｏｌ／Ｌとなるようにリン酸カルシウムを添加しＡ液とした。
24.3 mL of 0.1 mol / L citric acid and 25.7 mL of 0.2 mol / L disodium hydrogen phosphate were mixed to prepare a citrate phosphate buffer.
Calcium phosphate was added to this buffer solution so that the calcium phosphate concentration was 0.5 mol / L to obtain solution A.
非イオン性界面活性剤としてポリエチレングリコールモノオクチルフェニルエーテル（和光純薬社製）を水に添加し１０重量％の水溶液としたものをＢ液とした。
Ａ液とＢ液を等量混ぜ合わせｐＨ５の唾液の前処理液を作製した。
As a nonionic surfactant, polyethylene glycol monooctylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was added to water to give a 10% by weight aqueous solution.
A pretreatment liquid of pH 5 saliva was prepared by mixing equal amounts of A liquid and B liquid.
実施例１と同じ患者の唾液２５０μＬに唾液の前処理液を１００μＬ加えて混合したものを実施例１と同様の免疫クロマトグラフィー法を用いて試験を行った。抗体反応は全く確認できなかった。 A test was performed using the same immunochromatography method as in Example 1 by adding 100 μL of saliva pretreatment liquid to 250 μL of saliva of the same patient as in Example 1 and mixing them. No antibody reaction could be confirmed.
Claims (3)
するための唾液の前処理液であって、
非イオン性界面活性剤及び／または両性界面活性剤、
塩化ナトリウム，塩化カリウム，塩化カルシウム，塩化マグネシウム，硫酸マグネシウム
，硫酸マンガン，塩化アンモニウムからなる群より選ばれる１種または２種以上の物質、
緩衝作用付与物質、
及び水のみから構成され、
前記塩化ナトリウム，塩化カリウム，塩化カルシウム，塩化マグネシウム，硫酸マグネシウム，硫酸マンガン，塩化アンモニウムからなる群より選ばれる１種または２種以上の物質の濃度は唾液の前処理液中に０．２５〜５ｍｏｌ／Ｌであり、
ｐＨが３〜８である唾液の前処理液。 A pretreatment solution of saliva for pretreatment when identifying and quantifying periodontopathic bacteria in saliva by immunochromatography,
Nonionic surfactants and / or amphoteric surfactants,
One or more substances selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, magnesium sulfate, manganese sulfate, and ammonium chloride;
Buffering substance,
And only water ,
The concentration of one or more substances selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, magnesium sulfate, manganese sulfate, and ammonium chloride is 0.25 to 5 mol in the pretreatment solution of saliva. / L,
A pretreatment solution for saliva having a pH of 3 to 8.
メタン，塩酸−グリシン，グリシン−水酸化ナトリウム、クエン酸−リン水素二ナトリウ
ム、クエン酸−クエン酸ナトリウム，リン酸水素二ナトリウム−リン酸二水素ナトリウム
，炭酸ナトリウム−重炭酸ナトリウム，酢酸−酢酸ナトリウム，酒石酸−酒石酸ナトリウ
ム，酒石酸−トリス（ヒドロキシメチル）アミノメタンからなる物質の組み合わせからな
る群より選ばれる１種または２種以上である請求項１に記載の唾液の前処理液。 The buffering substances are hydrochloric acid-potassium chloride, hydrochloric acid-tris (hydroxymethyl) aminomethane, hydrochloric acid-glycine, glycine-sodium hydroxide, citric acid-disodium hydrogen phosphate, citric acid-sodium citrate, dihydrogen phosphate One or two selected from the group consisting of substances consisting of sodium-sodium dihydrogen phosphate, sodium carbonate-sodium bicarbonate, acetic acid-sodium acetate, tartaric acid-sodium tartrate, tartaric acid-tris (hydroxymethyl) aminomethane The pretreatment liquid for saliva according to claim 1, which is as described above.
アグリゲイティバクター・アクチノミセテムコミタンス、フゾバクテリウム・ヌクレアタ
ム、トレポネーマ・デンティコーラ、タンネレラ・フォーサイシアからなる群より選ばれ
る１種または２種以上である請求項１または２に記載の唾液の前処理液。 Periodontopathic bacteria include Porphyromonas gingivalis, Prevotella intermedia,
The pretreatment solution for saliva according to claim 1 or 2, wherein the pretreatment solution is one or more selected from the group consisting of Aggregatebacter actinomycetemcomitans, Fusobacterium nucleatum, Treponema denticola, and Tannerella fauciacia.
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