JPH1062402A - Quantifying method of sugar alcohol - Google Patents
Quantifying method of sugar alcoholInfo
- Publication number
- JPH1062402A JPH1062402A JP9180298A JP18029897A JPH1062402A JP H1062402 A JPH1062402 A JP H1062402A JP 9180298 A JP9180298 A JP 9180298A JP 18029897 A JP18029897 A JP 18029897A JP H1062402 A JPH1062402 A JP H1062402A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- column
- exchange resin
- sample
- resin
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 64
- 150000005846 sugar alcohols Chemical class 0.000 title claims abstract description 45
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 90
- 239000011347 resin Substances 0.000 claims abstract description 66
- 229920005989 resin Polymers 0.000 claims abstract description 66
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 63
- 239000000126 substance Substances 0.000 claims abstract description 47
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 230000002452 interceptive effect Effects 0.000 claims abstract description 20
- 239000002244 precipitate Substances 0.000 claims abstract description 15
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 210000004369 blood Anatomy 0.000 claims abstract description 4
- 239000008280 blood Substances 0.000 claims abstract description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 26
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 20
- 239000003456 ion exchange resin Substances 0.000 claims description 7
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 7
- MPCAJMNYNOGXPB-SLPGGIOYSA-N 1,5-anhydro-D-glucitol Chemical compound OC[C@H]1OC[C@H](O)[C@@H](O)[C@@H]1O MPCAJMNYNOGXPB-SLPGGIOYSA-N 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 4
- 239000003398 denaturant Substances 0.000 claims description 3
- 150000001450 anions Chemical class 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 abstract description 5
- 238000001556 precipitation Methods 0.000 abstract description 3
- 239000012530 fluid Substances 0.000 abstract 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 58
- 239000000243 solution Substances 0.000 description 38
- 210000002966 serum Anatomy 0.000 description 33
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 24
- 238000011088 calibration curve Methods 0.000 description 24
- 239000004327 boric acid Substances 0.000 description 19
- 239000003153 chemical reaction reagent Substances 0.000 description 19
- 239000007788 liquid Substances 0.000 description 19
- 238000005259 measurement Methods 0.000 description 18
- 238000004817 gas chromatography Methods 0.000 description 17
- 238000011002 quantification Methods 0.000 description 17
- 239000012153 distilled water Substances 0.000 description 16
- 239000003729 cation exchange resin Substances 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 239000000872 buffer Substances 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 108010001816 pyranose oxidase Proteins 0.000 description 12
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 11
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 11
- 239000005515 coenzyme Substances 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- 125000005619 boric acid group Chemical group 0.000 description 10
- 229960000367 inositol Drugs 0.000 description 9
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 9
- -1 proteins Chemical class 0.000 description 9
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 9
- 239000012086 standard solution Substances 0.000 description 9
- 101710088194 Dehydrogenase Proteins 0.000 description 8
- 108030001032 L-sorbose oxidases Proteins 0.000 description 8
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- 238000002835 absorbance Methods 0.000 description 7
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- 108090000854 Oxidoreductases Proteins 0.000 description 6
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- 239000007864 aqueous solution Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- ZTOJFFHGPLIVKC-YAFCTCPESA-N (2e)-3-ethyl-2-[(z)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound S\1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C/1=N/N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-YAFCTCPESA-N 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002523 gelfiltration Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 5
- 229920002451 polyvinyl alcohol Polymers 0.000 description 5
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 229940090047 auto-injector Drugs 0.000 description 4
- 125000002091 cationic group Chemical group 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000005342 ion exchange Methods 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 125000001453 quaternary ammonium group Chemical group 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 4
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IYLLULUTZPKQBW-UHFFFAOYSA-N Acrinol Chemical group CC(O)C(O)=O.C1=C(N)C=CC2=C(N)C3=CC(OCC)=CC=C3N=C21 IYLLULUTZPKQBW-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Natural products OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- 239000004372 Polyvinyl alcohol Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- BDAGIHXWWSANSR-UHFFFAOYSA-N formic acid Substances OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
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- 229920000193 polymethacrylate Polymers 0.000 description 3
- 150000003839 salts Chemical group 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 2
- GIAVHGFPMPSIFI-UHFFFAOYSA-N 3-hydroxy-2,4,6-triiodobenzoic acid Chemical compound OC(=O)C1=C(I)C=C(I)C(O)=C1I GIAVHGFPMPSIFI-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical group COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- XQXPVVBIMDBYFF-UHFFFAOYSA-N 4-hydroxyphenylacetic acid Chemical compound OC(=O)CC1=CC=C(O)C=C1 XQXPVVBIMDBYFF-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical group CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
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- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
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- 229920001778 nylon Polymers 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920013716 polyethylene resin Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000010349 pulsation Effects 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- XMVONEAAOPAGAO-UHFFFAOYSA-N sodium tungstate Chemical compound [Na+].[Na+].[O-][W]([O-])(=O)=O XMVONEAAOPAGAO-UHFFFAOYSA-N 0.000 description 1
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 1
- 239000012609 strong anion exchange resin Substances 0.000 description 1
- 125000000542 sulfonic acid group Chemical group 0.000 description 1
- GEVPIWPYWJZSPR-UHFFFAOYSA-N tcpo Chemical compound ClC1=CC(Cl)=CC(Cl)=C1OC(=O)C(=O)OC1=C(Cl)C=C(Cl)C=C1Cl GEVPIWPYWJZSPR-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は糖アルコールの定量
法、カラム及びキットに関する。１，５−アンヒドログ
ルシトール（以下ＡＧという）やミオイノシトール（以
下ＭＩという）などの糖アルコールは近年糖尿病のマー
カーとして注目を集めている物質である。The present invention relates to a method, a column and a kit for quantifying a sugar alcohol. Sugar alcohols such as 1,5-anhydroglucitol (hereinafter referred to as AG) and myo-inositol (hereinafter referred to as MI) are substances that have recently attracted attention as markers for diabetes.
【０００２】[0002]
【従来の技術】ヒト体液中の糖アルコールを定量する場
合、通常多量に含まれている糖類（特にグルコース）や
タンパク質などの定量に影響を与えるような干渉物質を
除去した試料を使用する。2. Description of the Related Art When quantifying a sugar alcohol in a human body fluid, a sample from which interfering substances which affect the quantification of saccharides (especially glucose) and proteins which are usually contained in a large amount is used.
【０００３】[0003]
【発明が解決しようとする課題】上記干渉物質の除去、
特にタンパク質の除去は従来遠心分離操作を必要とし、
煩雑であり、特に臨床応用を考える場合、その操作の簡
略化が望まれている。The removal of the above interfering substances,
In particular, protein removal conventionally requires centrifugation,
It is complicated, and simplification of the operation is desired especially when considering clinical applications.
【０００４】[0004]
【課題を解決するための手段】本発明は、 （１）糖アルコール、タンパク質、糖類を含有する検体
を、タンパク質除去能及び糖類除去能を有する塩基性陰
イオン交換樹脂を充填したカラムに通し、次いで通過液
中の糖アルコールを定量することを特徴とする糖アルコ
ールの定量方法。 （２）親水性ゲルろ過担体に４級アンモニウム基を導入
した強塩基性陰イオン交換樹脂及びホウ酸水溶液を充填
したタンパク質及び糖類除去用カラム （３）タンパク質除去能及び糖類除去能を有する塩基性
陰イオン交換樹脂及びホウ酸水溶液を充填したカラム、
糖アルコール定量用試薬及び検量線作製用糖アルコール
からなるキット （４）糖アルコール、タンパク質、糖類を含有する検体
に、油状沈澱を生じさせるタンパク質不溶化剤を添加
し、タンパク質を油状沈澱物として沈降させて上清液を
得、次いでその上清液をタンパク不溶化剤吸着樹脂及び
塩基性陰イオン交換樹脂を充填したカラムに通すか又
は、タンパク質沈澱剤及びタンパク質変性剤を添加
し、タンパク質を水不溶化物として析出させ、得られた
析出液を塩基性陰イオン交換樹脂を充填したカラムに通
し、次いで、得られた通過液中の糖アルコールを定量す
ることを特徴とする糖アルコールの定量方法及び （５）血中の１，５−アンヒドログルシトールを定量す
るにあたり、通液している干渉物質除去カラムとインジ
ェクターとバイオセンサーを備えた装置に多数の検体を
順次注入することを特徴とする１，５−アンヒドログル
シトールの定量法に関する。According to the present invention, (1) a sample containing a sugar alcohol, a protein, and a saccharide is passed through a column packed with a basic anion exchange resin having a protein removing ability and a sugar removing ability; Next, a method for quantifying a sugar alcohol, comprising quantifying the sugar alcohol in the passing solution. (2) A column for removing proteins and saccharides packed with a strongly basic anion exchange resin having a quaternary ammonium group introduced into a hydrophilic gel filtration carrier and an aqueous solution of boric acid (3) Basic having protein-removing ability and saccharide-removing ability A column packed with an anion exchange resin and an aqueous boric acid solution,
A kit comprising a sugar alcohol quantification reagent and a sugar alcohol for preparing a calibration curve (4) A protein insolubilizing agent that causes oily precipitation is added to a sample containing sugar alcohols, proteins, and saccharides to precipitate the protein as an oily precipitate. To obtain a supernatant, and then pass the supernatant through a column packed with a protein insolubilizing agent-adsorbing resin and a basic anion exchange resin, or add a protein precipitant and a protein denaturing agent to convert the protein into a water-insoluble substance. (5) a method for quantifying sugar alcohols, which comprises passing the resulting precipitate through a column filled with a basic anion exchange resin, and then quantifying the sugar alcohol in the resulting filtrate. ) To determine the amount of 1,5-anhydroglucitol in blood, use an interfering substance removal column, injector and Regarding determination of 1,5-anhydroglucitol, which comprises sequentially injecting a plurality of analytes in device with a sensor.
【０００５】[0005]
【発明の実施の形態】本発明で用いられる検体として
は、タンパク質や糖類の混入が予想され、かつ糖アルコ
ールを含有するものであれば特に制限は無く、例えば血
清、血漿、尿、髄液などの体液や植物、動物等の組織の
抽出液などがあげられる。糖アルコールとしては、糖類
の還元体である直鎖状の多価アルコールやこれらの化合
物が分子内で脱水閉環したアンヒドロ化合物及び環状の
多価アルコールが有る。これら糖アルコールのうちでＡ
ＧとＭＩは糖尿病の診断マーカーとして有用であり、糖
尿病患者の血清中のＡＧ濃度は著しく低下し、尿中のＭ
Ｉ濃度は上昇することが知られている。なお、糖類と
は、アルドース、ケトースなどの単糖類やこれらのオリ
ゴ糖類の意で、糖アルコールは含まない。BEST MODE FOR CARRYING OUT THE INVENTION The specimen used in the present invention is not particularly limited as long as it is expected to be contaminated with proteins and saccharides and contains a sugar alcohol. Examples thereof include serum, plasma, urine, and cerebrospinal fluid. And extracts of tissues of plants, animals and the like. Examples of the sugar alcohol include a linear polyhydric alcohol which is a reduced form of a saccharide, an anhydro compound in which these compounds are dehydrated and closed in a molecule, and a cyclic polyhydric alcohol. Of these sugar alcohols, A
G and MI are useful as diagnostic markers for diabetes, the concentration of AG in the serum of diabetic patients is significantly reduced, and the concentration of M in urine is reduced.
I concentrations are known to increase. The saccharide means a monosaccharide such as aldose or ketose or an oligosaccharide thereof, and does not include a sugar alcohol.
【０００６】本発明の第１の発明においてカラムに充填
されるタンパク質除去能及び糖類除去能を有する塩基性
陰イオン交換樹脂は糖アルコールの除去能を有さないも
ので、芳香環を有さない親水性の高分子担体、例えば親
水性ゲルろ過担体（分子ふるい作用を有する高分子ゲ
ル）に４級アンモニウム基を交換基として導入した強塩
基性の陰イオン交換樹脂が好ましい。親水性ゲルろ過担
体としては例えばデキストラン、アガロース、セルロー
ス、キトサンなどの多糖類系、ポリビニルアルコール
系、ポリエチレングリコール系、ポリアクリルアミド
系、ポリ（メタ）アクリレート系などの、好ましくはポ
リビニルアルコール系又はポリ（メタ）アクリレート系
の樹脂があげられる。又、４級アンモニウム基として
は、トリメチルアンモニウム基、トリエチルアンモニウ
ム基などのトリ低級（Ｃ1 −Ｃ4 ）アルキルアンモニウ
ム基、又、ヒドロキシエチルジメチルアンモニウム基等
のヒドロキシ低級（Ｃ1 −Ｃ4 ）アルキル・ジ低級（Ｃ
1 −Ｃ4 ）アンモニウム基があげられる。[0006] In the first invention of the present invention, the basic anion exchange resin having the ability to remove proteins and the ability to remove saccharides packed in a column does not have the ability to remove sugar alcohols and does not have an aromatic ring. A strongly basic anion exchange resin in which a quaternary ammonium group is introduced as an exchange group into a hydrophilic polymer carrier, for example, a hydrophilic gel filtration carrier (polymer gel having a molecular sieve action) is preferable. As the hydrophilic gel filtration carrier, for example, polysaccharides such as dextran, agarose, cellulose, and chitosan, polyvinyl alcohols, polyethylene glycols, polyacrylamides, poly (meth) acrylates, and the like, preferably polyvinyl alcohols or poly ( (Meth) acrylate-based resins. Examples of the quaternary ammonium group include a tri-lower (C 1 -C 4 ) alkyl ammonium group such as a trimethyl ammonium group and a triethyl ammonium group, and a hydroxy lower (C 1 -C 4 ) alkyl group such as a hydroxyethyl dimethyl ammonium group.・ Di lower grade (C
1 -C 4) ammonium group.
【０００７】これらの強塩基性樹脂のイオン型は、ＯＨ
形またはホウ酸形などの弱酸塩形として用いられる。好
ましい樹脂としてはMonoQ （フアルマシア社製）、Shod
exTM（昭和電工社製）、Nucleosil 100 −ＳＢ（ナーゲ
ル社製）、Partisil 10SAX （ワットマン社製）、Sepra
lyte SAX 、Bond Elute SAX（アナリテカル インター
ナショナル社製）、QAE-Toyopearl （東ソ社製）、QA-T
risacryl（ＩＢＦ社製）、Sepabeads FP-QA （三菱化成
製）、QAE-Sephadex、Q-Sepharose （ファルマシア社
製）、QAE-Cellulose （チッソ社製）などがある。より
好ましい樹脂としてはポリビニルアルコール系の樹脂で
あってトリエチルアンモニウム基を有するQAE-Toyopear
l があげられる。又、後記のバイオセンサーを用いる場
合はポリ（メタ）アクリレート系の樹脂であってトリメ
チルアンモニウム基を有するShodex TMもあげられる。The ionic form of these strong basic resins is OH
It is used as a weak acid salt form such as a form or a boric acid form. Preferred resins are MonoQ (Pharmacia), Shod
exTM (Showa Denko), Nucleosil 100-SB (Nagel), Partisil 10SAX (Whatman), Sepra
lyte SAX, Bond Elute SAX (manufactured by Analytical International), QAE-Toyopearl (manufactured by Toso), QA-T
risacryl (manufactured by IBF), Sepabeads FP-QA (manufactured by Mitsubishi Kasei), QAE-Sephadex, Q-Sepharose (manufactured by Pharmacia), QAE-Cellulose (manufactured by Chisso), and the like. More preferred resin is a polyvinyl alcohol-based resin having a triethylammonium group, QAE-Toyopear
l. When a biosensor described later is used, Shodex ™ which is a poly (meth) acrylate-based resin and has a trimethylammonium group can also be used.
【０００８】これらの樹脂の使用量は検体１ｍｌ当り１
ｍｌ以上好ましくは２〜６ｍｌ程度がよい。又、これら
の樹脂は目的に応じ２種以上併用してもよく、又、糖類
除去能が充分でない場合は後記の糖類除去能を有する樹
脂と併用してもよい。[0008] The amount of these resins used is 1 per ml of specimen.
ml or more, preferably about 2 to 6 ml. In addition, two or more of these resins may be used in combination depending on the purpose. If the saccharide removing ability is not sufficient, the resin may be used in combination with a resin having a saccharide removing ability described later.
【０００９】これらの樹脂はＯＨ形では安定性が無く、
長期間保存するとタンパク質除去能及び糖類除去能が低
下してくる。通常市販されるＣｌ形は安定であるが、ア
ルカリでＣｌを除去した後中性になるまで洗浄する必要
があるので、手間と時間がかかり好ましくない。本発明
者らは、ホウ酸形にすることにより安定性が向上し、使
用の際には水洗するだけでよいことを見い出した。即
ち、ＯＨ形又はホウ酸形の上記樹脂を樹脂が浸らない部
分が生じない程度の量のホウ酸水溶液、好ましくは０．
０５−１Ｍ濃度のホウ酸水溶液に浸しておくことにより
少なくとも１年間はその性能の低下を防ぐことができ
る。従って両者を充填したカラムを製造することによ
り、本発明の定量法が容易に実施しうる。また、これら
の樹脂は、イオン交換作用があるので、サンプル中にイ
オン性物質があると、イオン交換し流出液がアルカリ性
になるため、ｐＨ調整用に強カチオン交換樹脂を併用
し、例えばカラムの最下層に充填しても良く、例えばＡ
Ｇ50Ｗ−×８（バイオラッド社製）やAmberlite ＣＧ−
120 Type Ｉ（ローム＆ハース社製）などのスルフォン
酸を導入したイオン交換樹脂が用いられる。[0009] These resins are not stable in the OH form,
When stored for a long time, the ability to remove proteins and the ability to remove saccharides decrease. Usually, the commercially available Cl form is stable, but it is necessary to wash it until neutral after removing Cl with an alkali, so that it is troublesome and time-consuming, which is not preferable. The present inventors have found that the use of the boric acid form improves the stability, and it is only necessary to wash with water when used. That is, an amount of an aqueous boric acid solution, preferably an amount of an aqueous solution of boric acid which is small enough not to form a portion where the resin of the OH type or boric acid type is not immersed in the resin.
By immersing in an aqueous solution of boric acid having a concentration of 05-1M, the performance can be prevented from lowering for at least one year. Therefore, the quantification method of the present invention can be easily carried out by producing a column packed with both. In addition, since these resins have an ion exchange effect, if there is an ionic substance in the sample, ion exchange will be performed and the effluent will be alkaline. The lowermost layer may be filled, for example, A
G50W-x8 (manufactured by Bio-Rad) or Amberlite CG-
An ion exchange resin into which sulfonic acid is introduced, such as 120 Type I (manufactured by Rohm & Haas), is used.
【００１０】第１の発明を実施するには例えば次のよう
にすればよい。即ち、検体５０〜１００μｌをそのまま
上記カラムに通し、水０．５〜１．０ｍｌで該カラムを
２〜４回洗浄し、得られた通過液中に定量的に回収され
た糖アルコールをガスクロマトグラフィー法や酵素法な
どの常法により定量すればよい。なお、定量法によって
はタンパク質や糖類、特にタンパク質を完全に除去しな
くてもよい場合があり、この場合は、これらを定量に支
障のない程度に除去すればよい。The first invention may be implemented, for example, as follows. That is, 50 to 100 μl of a sample is passed through the column as it is, the column is washed 2 to 4 times with 0.5 to 1.0 ml of water, and the sugar alcohol quantitatively recovered in the obtained permeate is subjected to gas chromatography. It may be determined by a conventional method such as a photographic method or an enzyme method. In some cases, it is not necessary to completely remove proteins and saccharides, particularly proteins, depending on the quantification method. In this case, these may be removed to such an extent that quantification is not hindered.
【００１１】酵素法について説明すると、測定しようと
する糖アルコールと特異的に反応する酵素を用い、たと
えば、血清中のＡＧを測定する場合には、ピラノースオ
キシダーゼやＬ−ソルボースオキシダーゼ等の１，５−
ＡＧ酸化酵素が用いられ、ＡＧの酸化反応により発生し
た過酸化水素を、種々の方法により検出すればよい。ま
た、尿中のＭＩを測定する場合にはミオイノシトールデ
ヒドロゲナーゼが用いられ反応によって生じた補酵素β
−ニコチンアミドアデニンジヌクレオチド（ＮＡＤ）又
はβ−ニコチンアミドアデニンヌクレオチドリン酸（Ｎ
ＡＤＰ）のそれぞれの還元体ＮＡＤＨ又はＮＡＤＰＨを
検出すればよい。The enzymatic method will be described. An enzyme that specifically reacts with the sugar alcohol to be measured is used. For example, when AG in serum is measured, 1,5 such as pyranose oxidase or L-sorbose oxidase is used. −
AG oxidase may be used, and hydrogen peroxide generated by the oxidation reaction of AG may be detected by various methods. When measuring MI in urine, myo-inositol dehydrogenase is used, and coenzyme β generated by the reaction is used.
-Nicotinamide adenine dinucleotide (NAD) or β-nicotinamide adenine nucleotide phosphate (N
What is necessary is just to detect each reduced form NADH or NADPH of ADP).
【００１２】定量法をさらに詳しく説明すると、例えば
ＡＧの場合、次のようにすればよい。即ち、試料に電子
受容体及びピラノースオキシダーゼ又はＬ−ソルボース
オキシダーゼを試料１ｍｌ当り０．５〜１０単位、好ま
しくは１〜５単位添加し、４〜５０℃好ましくは２５〜
４０℃で、０．５〜３時間、好ましくは０．５〜１時間
インキュベートし次いで生成する過酸化水素量を測定
し、別に作成した検量線からＡＧ量を求めればよい。具
体的には次のとおりである。The quantification method will be described in more detail. For example, in the case of AG, the following method may be used. That is, an electron acceptor and pyranose oxidase or L-sorbose oxidase are added to a sample in an amount of 0.5 to 10 units, preferably 1 to 5 units, per 1 ml of the sample, and 4 to 50 ° C., preferably 25 to 50 units.
After incubating at 40 ° C. for 0.5 to 3 hours, preferably 0.5 to 1 hour, the amount of generated hydrogen peroxide is measured, and the amount of AG may be determined from a separately prepared calibration curve. The details are as follows.
【００１３】本発明で用いられる過酸化水素を検出する
方法としては、高感度に検出できる方法であれば、いず
れであっても良く、数多くの方法が利用できる。これら
のうちで最も一般的に用いられている方法は、ホースラ
ディッシュペルオキシダーゼ（ＨＲＰ）を触媒酵素とし
て、各種のＨＲＰ基質を過酸化水素で酸化するものであ
り、酸化反応の結果生成した色素、ケイ光物質や化学発
光をそれぞれ吸光度測定、ケイ光測定及び発光測定すれ
ば良い。色素を生成するＨＲＰの基質としては２，２′
−アジノビス（３−エチルベンゾチアゾリン−６−スル
ホン酸（ＡＢＴＳ）、ｏ−フェニレンジアミン（ＯＰ
Ｄ）、５−アミノサリチル酸（５−ＡＳ）、３，３′，
５，５′−テトラメチルベンジジン（ＴＭＢ）、４−ア
ミノアンチピリンと各種のトリンダー試薬などがある。
トリンダー試薬としては、フェノール、３−ヒドロキシ
−２，４，６−トリヨード安息香酸（ＨＴＩＢ）などの
フェノール誘導体、Ｎ−エチル−Ｎ−（２−ヒドロキシ
−３−スルホプロピル）−ｍ−トルイジン（ＴＯＯＳ）
や３，５−ジメトキシ−Ｎ−エチル−Ｎ−（２−ヒドロ
キシ−３−スルホプロピル）アニリン（ＤＡＯＳ）など
のアニリン誘導体がある。As the method for detecting hydrogen peroxide used in the present invention, any method can be used as long as it can be detected with high sensitivity, and many methods can be used. Of these, the most commonly used method is to oxidize various HRP substrates with hydrogen peroxide using horseradish peroxidase (HRP) as a catalytic enzyme. The light substance and the chemiluminescence may be measured by absorbance measurement, fluorescence measurement and luminescence measurement, respectively. 2,2 'is used as a substrate of HRP for producing a dye.
-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), o-phenylenediamine (OP
D), 5-aminosalicylic acid (5-AS), 3,3 ',
There are 5,5'-tetramethylbenzidine (TMB), 4-aminoantipyrine and various Trinder reagents.
As the Trinder reagent, phenol, phenol derivatives such as 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS) )
And aniline derivatives such as 3,5-dimethoxy-N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (DAOS).
【００１４】ケイ光物質を生成するＨＲＰの基質として
は、ｐ−ヒドロキシフェニル酢酸、３−（ｐ−ヒドロキ
シフェニル）プロピオン酸（ＨＰＰＡ）などがある。ま
た、化学発光するＨＲＰの基質としては、ルミノール、
イソルミノールなどが知られている。具体的には例えば
次のとおり、[0014] Examples of the HRP substrate for producing a fluorescent substance include p-hydroxyphenylacetic acid and 3- (p-hydroxyphenyl) propionic acid (HPPA). In addition, luminol,
Isorminol and the like are known. Specifically, for example:
【００１５】リン酸ナトリウム緩衝液（１／１５Ｍ，ｐ
Ｈ５．６）０．３ｍｌ、４ｍＭの２，２′−アジノビス
（３−エチルベンツチアゾリン−６−スルホン酸）（Ａ
ＢＴＳ）と１２単位／ｍｌのホースラディッシュペルオ
キシダーゼを含む発色液０．５ｍｌ、２５単位／ｍｌの
ピラノースオキシダーゼ又はＬ−ソルボースオキシダー
ゼ溶液０．１ｍｌ及びＡＧ溶液０．１ｍｌを容器に入
れ、２５°で１時間反応させた後、４２０ｎｍにおける
吸光度を測定する。既知濃度のＡＧ溶液で検量線を作成
しておき試料の吸光度よりＡＧの濃度を算出する。A sodium phosphate buffer (1/15 M, p
H5.6) 0.3 ml, 4 mM 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (A
BTS) and 0.5 ml of a color developing solution containing 12 units / ml of horseradish peroxidase, 0.1 ml of a 25 units / ml pyranose oxidase or L-sorbose oxidase solution, and 0.1 ml of an AG solution were placed in a container, and 25 ° After reacting for an hour, the absorbance at 420 nm is measured. A calibration curve is prepared with an AG solution having a known concentration, and the AG concentration is calculated from the absorbance of the sample.
【００１６】ＨＲＰなしに化学発光で検出する方法もい
くつか知られている。たとえば、フェリシアンイオン存
在下に過酸化水素でルミノールを発光させる方法、金属
イオン存在下に過酸化水素でルシゲニンを発光させる方
法、ビス（２，４，６−トリクロルフェニル）オキザレ
ートの様なアリルシュウ酸エステル類の化合物をケイ光
物質存在下に過酸化水素と反応させ、シュウ酸エステル
の分解エネルギーでケイ光物質を励起させ発光させる方
法などが知られている。さらに過酸化水素を直接検出す
る方法として、過酸化水素電極を用いてもよい。Several methods for detecting chemiluminescence without HRP are also known. For example, a method of emitting luminol with hydrogen peroxide in the presence of ferricyan ions, a method of emitting lucigenin with hydrogen peroxide in the presence of metal ions, an allyl oxalic acid such as bis (2,4,6-trichlorophenyl) oxalate There is known a method in which an ester compound is reacted with hydrogen peroxide in the presence of a fluorescent substance to excite the fluorescent substance with the decomposition energy of the oxalate to emit light. Further, as a method of directly detecting hydrogen peroxide, a hydrogen peroxide electrode may be used.
【００１７】又、たとえば、ＭＩの場合、次のようにす
ればよい。即ち、カラム通過液に補酵素及びミオイノシ
トールデヒドロゲナーゼを試料１ｍｌ当り０．１〜１０
単位、好ましくは０．５〜５単位添加し、４〜５０℃、
好ましくは２５〜４０℃で０．５〜３時間、好ましくは
０．５〜２時間インキュベートし、次いで生成する補酵
素の還元体を測定し、別に作成した検量線からＭＩ量を
求めればよい。In the case of MI, for example, the following may be performed. That is, the co-enzyme and myo-inositol dehydrogenase were added to the column-passing liquid in an amount of 0.1 to 10 per ml of the sample.
Unit, preferably 0.5 to 5 units, 4 to 50 ° C,
It is preferable to incubate at 25 to 40 ° C. for 0.5 to 3 hours, preferably 0.5 to 2 hours, measure the reduced form of the coenzyme formed, and determine the MI amount from a separately prepared calibration curve.
【００１８】本発明で用いられる補酵素の還元体、たと
えばＮＡＤＨやＮＡＤＰＨを検出する方法としては、高
感度に検出できる方法であればいずれであっても良く、
数多くの方法が利用できる。これらのうちで、最も一般
的に用いられている方法は、これらの還元体そのものの
吸光度もしくはケイ光強度を測定するものである。さら
には、生成した補酵素の還元体をフェメジンメトサルフ
ェート、１−メトキシ−５−メチルフェナジウムメチル
サルフェート、メチレンブルーなどの電子伝達体の存在
下に空気酸化し、発生した過酸化水素を前述の方法によ
り検出することもできる。具体的には、例えば次のとお
り。The method for detecting the reduced form of the coenzyme used in the present invention, for example, NADH or NADPH, may be any method as long as it can be detected with high sensitivity.
Numerous methods are available. Of these, the most commonly used method is to measure the absorbance or fluorescence intensity of these reduced forms themselves. Furthermore, the reduced form of the produced coenzyme is air-oxidized in the presence of an electron carrier such as femedin methosulfate, 1-methoxy-5-methylphenadium methyl sulfate, and methylene blue, and the generated hydrogen peroxide is treated as described above. Can also be detected. Specifically, for example, as follows.
【００１９】１４０ｍＭの硫安を含む４０ｍＭ炭酸ナト
リウム緩衝液（ｐＨ９．０）１．０ｍｌ、７ｍＭ ＮＡ
Ｄ溶液０．２ｍｌ、１０単位／ｍｌのミオイノシトール
デヒドロゲナーゼ溶液０．２ｍｌ、蒸留水０．４ｍｌ及
びＭＩ溶液０．２ｍｌを容器に入れ、２５℃で２時間反
応させた後、励起波長３６５ｎｍ、検出波長４５０ｎｍ
におけるケイ光強度を測定する。既知濃度のＭＩ溶液で
検量線を作成しておき、試料のケイ光強度よりＭＩの濃
度を算出する。1.0 ml of 40 mM sodium carbonate buffer (pH 9.0) containing 140 mM ammonium sulfate, 7 mM NA
0.2 ml of D solution, 0.2 ml of 10 units / ml myo-inositol dehydrogenase solution, 0.4 ml of distilled water and 0.2 ml of MI solution were placed in a container, reacted at 25 ° C. for 2 hours, and then excited at 365 nm. 450nm wavelength
The fluorescence intensity at is measured. A calibration curve is prepared with an MI solution having a known concentration, and the MI concentration is calculated from the fluorescence intensity of the sample.
【００２０】第３の発明におけるキットは上記したタン
パク質除去能及び糖類除去能を有する塩基性陰イオン交
換樹脂及びホウ酸水溶液を充填したカラム、糖アルコー
ル定量用酵素及び検出用試薬からなる糖アルコール定量
用試薬及び検量線作成用糖アルコールをワンセットした
ものである。カラムは除タンパク、、除糖しうる親水性
ゲルろ過担体を基材とした強塩基性の陰イオン交換樹脂
を上層に陽イオン交換樹脂を下層に充填したディスポー
ザブルなミニカラムである。このカラムに充填する樹脂
の１回当りの使用量は例えば次のとおり。The kit according to the third invention comprises a column packed with a basic anion exchange resin having the above-mentioned protein-removing ability and saccharide-removing ability and an aqueous boric acid solution, a sugar alcohol quantification enzyme comprising a sugar alcohol quantification enzyme and a detection reagent. And one set of sugar alcohol for preparing a calibration curve. The column is a disposable mini-column in which a strongly basic anion exchange resin based on a hydrophilic gel filtration carrier capable of removing proteins and sugar is filled in an upper layer and a cation exchange resin is filled in a lower layer. The amount of the resin to be packed in this column per use is as follows, for example.
【００２１】 親水性ゲルろ過担体より成る塩基性 陰イオン交換樹脂（ＯＨ形又はホウ酸形） ０．１〜２．０ｍｌ ホウ酸 〜 ｍｌ 陽イオン交換樹脂 ０．０１〜０．５ｍｌ Ｓｕｂ Ｔｏｔａｌ ０．５〜２．０ｍｌ ホウ酸 ０．５〜 １０ｍｌ 全量で １〜 １２ｍｌBasic anion exchange resin (OH form or boric acid form) consisting of hydrophilic gel filtration carrier 0.1 to 2.0 ml boric acid to ml cation exchange resin 0.01 to 0.5 ml Sub Total 0. 5 to 2.0 ml Boric acid 0.5 to 10 ml 1 to 12 ml in total volume
【００２２】糖アルコール定量用酵素としては、たとえ
ば、ＡＧの場合、ＡＧの定量に直接使用しうる酵素であ
れば特に制限はなく、例えば前記のピラノースオキシダ
ーゼやＬ−ソルボースオキシダーゼなどがあげられる。
また、たとえばＭＩの場合ＭＩの定量に直接使用しうる
酵素であれば特に制限はなく、例えば前記のミオイノシ
トールデヒドロゲナーゼがあげられる。The enzyme for sugar alcohol quantification is not particularly limited in the case of AG, for example, as long as it is an enzyme which can be directly used for quantification of AG, and examples thereof include the aforementioned pyranose oxidase and L-sorbose oxidase.
Further, for example, in the case of MI, there is no particular limitation as long as the enzyme can be used directly for the quantification of MI, and examples thereof include the aforementioned myo-inositol dehydrogenase.
【００２３】糖アルコール定量用試薬としては、発生す
る過酸化水素量を指標とする場合、パーオキシダーゼも
しくはパーオキシダーゼ様活性物質と発色基質もしくは
発色剤およびカップラー、パーオキシダーゼとケイ光基
質、パーオキシダーゼと発光基質、フェリシアンイオン
と発光基質の組み合せなどが例示される。これらの試薬
の具体例は前記「過酸化水素を検出する方法」の記載よ
り明らかである。When the amount of hydrogen peroxide generated is used as an index, the reagent for quantifying sugar alcohols may be a peroxidase or peroxidase-like active substance, a chromogenic substrate or a chromogenic agent, a coupler, a peroxidase and a fluorescent substrate, and a peroxidase. Examples include a luminescent substrate, a combination of a ferricyan ion and a luminescent substrate, and the like. Specific examples of these reagents are apparent from the description of the above-mentioned "method for detecting hydrogen peroxide".
【００２４】又、補酵素の還元体たとえばＮＡＤＨやＮ
ＡＤＰＨの量を指標とする場合、補酵素自身とし、その
もの自身の吸光度もしくはケイ光強度を測定する。一
方、補酵素の還元体を電子伝達体を介して過酸化水素に
変換すれば、前記の過酸化水素検出試薬が使用できる。Also, coenzyme reductants such as NADH and N
When the amount of ADPH is used as an index, the coenzyme itself is used, and the absorbance or fluorescence intensity of the coenzyme itself is measured. On the other hand, if the reduced form of the coenzyme is converted into hydrogen peroxide via an electron carrier, the above-mentioned reagent for detecting hydrogen peroxide can be used.
【００２５】キット中の糖アルコール定量用の酵素の量
は１００検体用、３００検体用など測定し得る試料の数
により異なるが、１検体当り、たとえばＡＧを定量する
場合０．５〜１０単位程度、ＭＩを定量する場合０．１
〜５単位程度を加えればよい。又、定量用試薬について
は、過酸化水素検出用試薬としてＨＲＰとＡＢＴＳを用
いる場合、ＨＲＰ１検体当り、０．０１〜０．１単位、
ＡＢＴＳは１〜２０μＭになるようにキット中に入れ
る。The amount of the enzyme for quantifying the sugar alcohol in the kit varies depending on the number of measurable samples, such as for 100 samples and 300 samples, but about 0.5 to 10 units per sample, for example, when quantifying AG. , When quantifying MI 0.1
What is necessary is just to add about 5 units. When HRP and ABTS are used as reagents for detecting hydrogen peroxide, 0.01 to 0.1 units per HRP sample,
ABTS is included in the kit at 1 to 20 μM.
【００２６】補酵素の還元体の検出方法として、そのも
ののケイ光強度を測定する場合、補酵素は１検体当り、
０．２〜１０μＭになるようにキットの中に入れる。ま
た、この他、検量線作成用試料として、たとえばＡＧ又
はＭＩの場合、それぞれの１００〜１０００μＭをキッ
トに組み込むのがよい。なお、１，５−ＡＧ定量用酵素
及び検出用試薬は、全てを混合して単一の試薬としても
よく、相互に干渉する成分が存在する場合には、各成分
を適宜な組み合せとなる様に分割してもよい。また、こ
れらは、溶液状、もしくは粉末状試薬として調製しても
よく、さらにこれらを濾紙もしくはフィルムなどの適当
な支持体に含有させ試験紙もしくは分析用フィルムとし
て調製してもよい。As a method for detecting a reduced form of a coenzyme, when the fluorescence intensity of the coenzyme is measured, the coenzyme is used per sample.
Place in the kit to be 0.2-10 μM. In addition, in the case of, for example, AG or MI as a sample for preparing a calibration curve, it is preferable to incorporate 100 to 1000 μM of each into a kit. In addition, the enzyme for 1,5-AG quantification and the reagent for detection may be mixed to form a single reagent. If there are components that interfere with each other, the components may be appropriately combined. May be divided. These may be prepared as a solution or a powdery reagent, or may be contained in a suitable support such as filter paper or film to prepare a test paper or a film for analysis.
【００２７】次に第５の発明であるバイオセンサーを用
いる自動定量法につき説明する。この方法は干渉物質除
去カラムに水あるいはほう酸緩衝液等を通液していると
ころにサンプルを注入し、サンプル中の干渉物質を吸着
除去し、ＡＧを含む通過液を直接バイオセンサーで検出
する方法である。したがって、本発明に用いる装置は、
基本的には、検体を注入するためのインジェクターとポ
ンプ等を用いることにより通液している干渉物質除去カ
ラムとＡＧを検出するためのバイオセンサーから成って
いる。Next, an automatic quantification method using a biosensor according to the fifth invention will be described. In this method, a sample is injected into a place where water or borate buffer is passed through an interference substance removal column, the interference substance in the sample is adsorbed and removed, and the passing liquid containing AG is directly detected by a biosensor. It is. Therefore, the device used in the present invention is:
Basically, it is composed of an interfering substance removal column through which liquid is passed by using an injector and a pump for injecting a specimen, and a biosensor for detecting AG.
【００２８】本発明で用いられるバイオセンサーとして
は、例えば１，５−ＡＧ酸化酵素を固定化した膜を過酸
化水素電極表面に装着したフロ・セル型検出器が挙げら
れる。フロ・セル中を通過しているＡＧは、過酸化水素
電極表面のＡＧ酸化酵素によって酸化され、同時に過酸
化水素を生成し、これを過酸化水素電極によって検出
し、ＡＧを定量するセンサーである。また、膜型の固定
化酵素ではなく、小カラムに固定化酵素を充填したカラ
ムを用いたリアクター型のセンサーを用いても同時にＡ
Ｇを定量することができる。更には、このリアクター型
の場合、ＡＧは過酸化水素に変換されて出てくるので、
過酸化水素電極の代わりに、過酸化水素を検出できる電
気化学検出器を用いたバイオセンサーを使用することも
できる。The biosensor used in the present invention includes, for example, a flow cell type detector in which a membrane on which 1,5-AG oxidase is immobilized is mounted on the surface of a hydrogen peroxide electrode. AG passing through the flow cell is oxidized by the AG oxidase on the surface of the hydrogen peroxide electrode, and simultaneously produces hydrogen peroxide, which is detected by the hydrogen peroxide electrode and is a sensor for quantifying AG. . In addition, even if a reactor-type sensor using a small column filled with immobilized enzyme instead of a membrane-type immobilized enzyme is used, A
G can be quantified. Furthermore, in the case of this reactor type, AG is converted into hydrogen peroxide and comes out,
Instead of the hydrogen peroxide electrode, a biosensor using an electrochemical detector capable of detecting hydrogen peroxide can also be used.
【００２９】１，５−ＡＧ酸化酵素の固定化の方法は、
一般的に行われている酵素固定化法例えば吸着法、架線
法、共有結合法のいずれの方法を採用しても良いが、基
質であるＡＧおよび過酸化水素の透過性の良い膜を得る
方法を採用するのが好ましい。リアクター型の場合、通
液性の良いものを採用するのが好ましい。The method for immobilizing 1,5-AG oxidase is as follows.
Any of the commonly used enzyme immobilization methods such as an adsorption method, an overhead wire method, and a covalent bonding method may be employed, but a method for obtaining a membrane having good permeability for the substrate AG and hydrogen peroxide. It is preferred to employ In the case of a reactor type, it is preferable to employ a liquid having good liquid permeability.
【００３０】過酸化水素電極を用いたバイオセンサーで
は、電気化学的に酸化還元反応を検出しているため、還
元性あるいは酸化性のある物質では、シグナルが出て過
酸化水素と同様に検出してしまうため、検体中にこのよ
うな物質が共存しているとＡＧだけを検出することがで
きない。特に血液中に存在するアスコルビン酸、尿酸等
によって、妨害されるため、ＡＧのみを測定することが
できない。そこで一般的にはこれらの物質の透過性のな
い保護膜を装置して防いでいる。ところが、本発明によ
る方法では、前記の強塩基性陰イオン交換樹脂を充填し
た干渉物質除去カラムを用いることで、これらの妨害物
質を吸着除去することができ、該カラム流出液を直接バ
イオセンサーで測定することが可能となった。なお、バ
イオセンサーでの測定には、ほとんと影響が無いが検体
中には多量のタンパクがあるため、糖類を吸着する官能
基をタンパクが覆ってしまって処理能力を著しく低下さ
せることがあるので、充填剤の素材には、注意を要す
る。好ましくは、疎水性の強い芳香環を持たない比較的
親水性の素材のアクリル系、親水性素材のポリビニルア
ルコール系の素材に第４級アンモニウム基を導入した硬
質の樹脂が良い。また、これらの樹脂のＯＨ形あるいは
ほう酸形の樹脂では、前述したようにバイオセンサーの
測定を妨害する血中成分をも除去するので、バイオセン
サーにて連続的に検出するには最適の干渉物質除去カラ
ムとなっている。また、これらの樹脂は、ンオン交換作
用があるので、サンプル中にイオン性物質があると、イ
オン交換し流出液がアルカリ性になるため、ｐＨ調整用
に強カチオン交換樹脂を併用して充填しても良く、スル
フォン酸を導入したイオン交換樹脂が用いられる。In a biosensor using a hydrogen peroxide electrode, an oxidation-reduction reaction is electrochemically detected, so that a substance having a reducing or oxidizing property produces a signal and is detected similarly to hydrogen peroxide. Therefore, if such a substance coexists in the sample, only AG cannot be detected. In particular, it is not possible to measure only AG because it is disturbed by ascorbic acid, uric acid, etc. present in blood. Therefore, in general, a protective film having no permeability to these substances is provided to prevent such substances. However, in the method according to the present invention, these interfering substances can be adsorbed and removed by using the interfering substance removal column packed with the strong basic anion exchange resin. It became possible to measure. The measurement with a biosensor has almost no effect, but since there is a large amount of protein in the sample, the functional group that adsorbs saccharides may be covered by the protein and the processing capacity may be significantly reduced. Attention must be paid to the material of the filler. Preferably, a hard resin in which a quaternary ammonium group is introduced into a relatively hydrophilic material such as an acrylic material having no strong aromatic ring and a polyvinyl alcohol material as a hydrophilic material is preferable. In addition, the OH-type or boric acid-type resins of these resins also remove blood components that interfere with the measurement of the biosensor as described above. It is a removal column. In addition, since these resins have an on-exchange effect, if there is an ionic substance in the sample, ion exchange will occur and the effluent will be alkaline. Alternatively, an ion exchange resin into which sulfonic acid is introduced is used.
【００３１】カラムの通液速度は、バイオセンサー部分
の接触時間と関係し、接触時間によって感度と測定時間
が変わるので慎重に選ぶ必要があるが、干渉物質除去カ
ラムの内径が４〜１００ｍｍφの場合０．１から５．０
ｍｌ／ｍｉｎ程度の流速が好ましい。このときの通液
は、圧力差を用いて流下させても良いが、流速のコント
ロールが難しいので、ポンプを使用したほうが良い。使
用するポンプは脈動の少ないものが望ましく、シリンダ
ー型、プランジャー型が優れている。インジェイターは
シリンダータイプ、固定ループタイプのどちらでも構わ
ないが、低いＡＧ濃度の検体の再現性を良くするために
は、変動の少ないインジェクターを使用したほうが良
い。全自動のシステムでは、オートインジェクターを使
用するのは言うまでもない。また、サンプルの注入量
は、測定感度にもよるが、少ないほうが、干渉物質除去
カラムの耐久性にかかわるので良く、５〜５０μｌ程度
が好ましい。本発明で用いるサンプル（検体）として
は、除タンパクをしていない血清又は血漿をそのまま用
いることが出来るが、後記の如く血清又は血漿にタンパ
ク不溶化剤を添加してタンパクを水不溶化したものをサ
ンプルとして用いてもよい。The flow rate of the column is related to the contact time of the biosensor portion, and the sensitivity and the measurement time depend on the contact time. Therefore, it is necessary to carefully select the flow rate. However, when the inner diameter of the column for removing an interfering substance is 4 to 100 mmφ, 0.1 to 5.0
A flow rate of the order of ml / min is preferred. At this time, the liquid may be caused to flow down using a pressure difference, but it is difficult to control the flow rate, so it is better to use a pump. It is desirable that the pump used has little pulsation, and the cylinder type and the plunger type are excellent. The injector may be either a cylinder type or a fixed loop type. However, in order to improve the reproducibility of a sample having a low AG concentration, it is preferable to use an injector with little fluctuation. It goes without saying that a fully automatic system uses an auto injector. The injection amount of the sample depends on the measurement sensitivity, but the smaller the amount, the better the influence on the durability of the column for removing an interfering substance, and is preferably about 5 to 50 μl. As the sample (specimen) used in the present invention, serum or plasma which has not been deproteinized can be used as it is, but as described below, a sample obtained by adding a protein insolubilizing agent to serum or plasma to insolubilize the protein in water is used as a sample. May be used.
【００３２】カラムの通液には、水あるいはほう酸緩衝
液等が用いられているが、ほう酸緩衝液ではその濃度が
高いと糖類を吸着しない場合があり、低い濃度のほう酸
緩衝液が望ましく、０．２Ｍ以下、好ましくは０．１Ｍ
以下、さらに好ましくは０．００１−０．０５Ｍの濃度
が好ましい。この場合ほう酸緩衝液のｐＨは、干渉物質
除去カラムの充填剤の種類によって変える必要があり、
強カチオン交換樹脂を併用した場合は、陽イオンが吸着
するため、ｐＨを上げたほう酸緩衝液は好ましくなく、
むしろほう酸単独のほうが好ましい。強アニオン交換樹
脂単独で使用した場合には、陽イオンは通過するのでい
ずれのｐＨでも良く、バイオセンサーの酵素の至適ｐ
Ｈ、例えばｐＨ５〜９を採用することができる。水ある
いは低濃度のほう酸では、緩衝作用が小さいため、サン
プル中のイオン性物質が、干渉物質除去カラムでイオン
交換されｐＨが変動する。そのため、酵素反応の至適ｐ
Ｈを維持することができず、バイオセンサーの酵素膜の
耐久性を悪くしたり、ノイズやショックの原因となる場
合がある。そこで、至適ｐＨを維持するため干渉物質除
去カラムの後で、緩衝作用の強い緩衝液を混合すること
は、バイオセンサーの耐久性に良い結果を与える。ここ
で用いられる緩衝液は、ＡＧ酸化酵素の至適ｐＨ、例え
ばｐＨ５〜９を維持できる緩衝液であれば、何でも良
く、例えばリン酸緩衝液が挙げられる。Water or borate buffer or the like is used to pass through the column. If the concentration of borate buffer is high, saccharides may not be adsorbed. If the concentration is high, a low concentration of borate buffer is desirable. .2M or less, preferably 0.1M
Hereinafter, the concentration is more preferably 0.001 to 0.05M. In this case, the pH of the borate buffer needs to be changed depending on the type of packing material in the column for removing the interfering substance.
When a strong cation exchange resin is used in combination, cations are adsorbed, so a borate buffer with an increased pH is not preferable.
Rather, boric acid alone is preferred. When a strong anion exchange resin is used alone, any pH may be used since cations pass through, and the optimal pH of the biosensor enzyme is sufficient.
H, for example, pH 5-9 can be employed. With water or low-concentration boric acid, the buffering action is small, so that the ionic substance in the sample is ion-exchanged by the interference substance removal column and the pH fluctuates. Therefore, the optimal p
H cannot be maintained, which may degrade the durability of the enzyme membrane of the biosensor, or cause noise or shock. Thus, mixing a buffer with a strong buffering action after the interfering substance removal column to maintain the optimum pH gives good results to the durability of the biosensor. The buffer used here may be any buffer as long as it can maintain the optimum pH of the AG oxidase, for example, pH 5 to 9, such as a phosphate buffer.
【００３３】本発明による方法の好ましい測定法を挙げ
ると、オートインジェクターと干渉物質除去カラムと
１，５−ＡＧ酸化酵素固定化膜を装着した過酸化水素電
極型のバイオセンサーに蒸留水あるいは低濃度のほう酸
緩衝液を流し、干渉物質除去カラムとバイオセンサーの
間にミキシングジョイントを設けリン酸緩衝液を混合
し、ミキシングコイルを経てバイオセンサーに接続して
いるものである。バイオセンサーは記録計あるいはデー
タ処理機につながり、過酸化水素電極からのシグナルを
ピークとし、面積あるいは高さとして定量する。オート
インジェクターから標準ＡＧ及びサンプルである血清あ
るいは血漿を注入し、干渉物質除去カラムの通過液中の
ＡＧのみがバイオセンサーで過酸化水素の量として定量
される。標準ＡＧから検量線を作成し、その検量線を元
にサンプル中のＡＧが定量されてくる。The preferred method of the method according to the present invention is as follows. A hydrogen peroxide electrode type biosensor equipped with an autoinjector, an interfering substance removal column and a 1,5-AG oxidase immobilized membrane is provided with distilled water or low-concentration. And a mixing joint is provided between the interfering substance removing column and the biosensor to mix the phosphate buffer, and the mixture is connected to the biosensor via a mixing coil. The biosensor is connected to a recorder or a data processor, and the signal from the hydrogen peroxide electrode is peaked and quantified as an area or height. A standard AG and serum or plasma as a sample are injected from the auto injector, and only the AG in the liquid passing through the column for removing the interfering substance is quantified by the biosensor as the amount of hydrogen peroxide. A calibration curve is created from the standard AG, and AG in the sample is quantified based on the calibration curve.
【００３４】次に第４の発明について説明する。第４の
発明ので用いられている油状沈澱を生じさせるタンパ
ク不溶化剤とは、該タンパク不溶化剤を加えることによ
り、タンパク質が油状沈澱物として析出し、遠心分離操
作をすることなしに除タンパク上清を得ることができる
様にするものである。たとえば、アクリノールなどが有
る。その使用量は、検体１ｍｌ当り２〜５０ｍｇ、好ま
しくは５〜２０ｍｇ程度であり、水溶液として検体に添
加することが好ましい。Next, the fourth invention will be described. The protein insolubilizing agent used to produce an oily precipitate used in the fourth invention is a protein insoluble agent added to precipitate a protein as an oily precipitate and remove the protein supernatant without centrifugation. Is to be obtained. For example, there is acrinol. The used amount is 2 to 50 mg, preferably about 5 to 20 mg per 1 ml of the sample, and it is preferable to add the solution as an aqueous solution to the sample.
【００３５】また、タンパク不溶化剤吸着樹脂とは、除
タンパクのために過剰に加えられたタンパク不溶化剤を
吸着し得るものであれば何でもよく、アクリノールを用
いた場合には、ダイヤイオンＨＰ−２０ＳＳ（三菱化成
製）などの疎水性樹脂が好ましい。これらのタンパク不
溶化剤吸着樹脂は塩基性陰イオン交換樹脂の上層部に添
加すればよく、一回のクロマト操作により、タンパク不
溶化剤の除去と糖類の除去の両方を同時に行うことがで
きる。The resin for adsorbing a protein insolubilizing agent may be any resin capable of adsorbing a protein insolubilizing agent excessively added for deproteinization. If acrinol is used, Diaion HP-20SS is used. Hydrophobic resins such as (manufactured by Mitsubishi Kasei) are preferred. These protein-insolubilizing agent-adsorbing resins may be added to the upper layer of the basic anion-exchange resin, and both the removal of the protein-insolubilizing agent and the removal of saccharides can be performed simultaneously by one chromatographic operation.
【００３６】塩基性陰イオン交換樹脂は主として糖類を
除去するものであり、通常ＯＨ形又はホウ酸形として用
いられる。陰イオン交換樹脂のＯＨ形を用いる方法で
は、検体を強塩基性樹脂のＯＨ形にゆっくりと通し、糖
類を吸着除去するものである。好ましい強塩基樹脂は、
４級アンモニウム塩を交換基とする樹脂で、強塩基性の
トリメチルアミノ基（Ｉ型）やヒドロキシエチルジメチ
ルアミノ基（ＩＩ基）を有する陰イオン交換樹脂、トリ
エチルアミノ基を有する陰イオン交換樹脂（ＱＡＥ−樹
脂）などが有る。また、この方法では、処理液の樹脂中
の通過速度の影響を受けるため、樹脂の粒度を細かく
（２００〜４００メッシュ）し、ゆっくり通過させるの
が好ましい。また、処理液を中和するために、さらに陽
イオン交換樹脂で処理しても良い。The basic anion exchange resin mainly removes saccharides and is usually used as an OH form or a boric acid form. In the method using the OH form of the anion exchange resin, the sample is slowly passed through the OH form of the strongly basic resin to adsorb and remove saccharides. Preferred strong base resins are
A resin having a quaternary ammonium salt as an exchange group, an anion exchange resin having a strongly basic trimethylamino group (type I) or a hydroxyethyldimethylamino group (group II), or an anion exchange resin having a triethylamino group ( QAE-resin). Further, in this method, since the processing liquid is affected by the passing speed of the resin, it is preferable that the particle size of the resin is fine (200 to 400 mesh) and the resin is passed slowly. Further, in order to neutralize the treatment solution, the treatment solution may be further treated with a cation exchange resin.
【００３７】陽イオン交換樹脂とは、各種の陰イオン性
樹脂のＨ形である。陰イオン性樹脂としては、強酸性の
陽イオン交換樹脂から弱酸性の陽イオン交換樹脂まで、
あらゆるタイプの陽イオン交換樹脂を包含する。特に好
ましくは、スルホン酸基を有する強酸性陽イオン交換樹
脂のＨ形などがある。The cation exchange resin is an H type of various anionic resins. As anionic resin, from strongly acidic cation exchange resin to weakly acidic cation exchange resin,
Includes all types of cation exchange resins. Particularly preferred is H-form of a strongly acidic cation exchange resin having a sulfonic acid group.
【００３８】陰イオン交換樹脂のホウ酸形を用いる方法
では糖をホウ酸との複合体として吸着除去するものであ
るが、ホウ酸形の陰イオン交換樹脂であれば特に制限は
なく、強塩基性の陰イオン交換樹脂から弱塩基性の陰イ
オン交換樹脂まであらゆるタイプの陰イオン交換樹脂を
包含する。特に好ましくは、強塩基性のトリメチルアミ
ノ基（Ｉ型）を有する陰イオン交換樹脂のホウ酸塩形、
強塩基性のヒドロキシエチルジメチルアミノ基（ＩＩ
型）を有する陰イオン交換樹脂のホウ酸塩形、トリエチ
ルアミノ基を有する陰イオン交換樹脂（ＱＡＥ−樹脂）
のホウ酸塩形、中塩基性でジメチルアミノ基とヒドロキ
シエチルジメチルアミノ基の２種類のイオン交換基を有
する樹脂（バイオラッド社製、バイオレックス５）のホ
ウ酸塩形などが有る。また、検体の処理により脱落して
来るホウ酸イオンを再吸着するため、さらに陰イオン交
換樹脂で処理しても良い。さらに、処理液を中和するた
めに、陽イオン交換樹脂による処理を追加しても良い。In the method using the boric acid form of the anion exchange resin, the sugar is adsorbed and removed as a complex with boric acid. However, there is no particular limitation as long as the boric acid form of the anion exchange resin is used. It includes all types of anion exchange resins, from neutral anion exchange resins to weakly basic anion exchange resins. Particularly preferably, a borate form of an anion exchange resin having a strongly basic trimethylamino group (form I),
Strongly basic hydroxyethyldimethylamino group (II
Exchange resin having a triethylamino group (QAE-resin)
And a borate form of a resin (BioRex, manufactured by Bio-Rad Co., Ltd.) having two basic types of ion-exchange groups of a dimethylamino group and a hydroxyethyldimethylamino group. Further, in order to re-adsorb borate ions coming off by the treatment of the sample, the sample may be further treated with an anion exchange resin. Further, a treatment with a cation exchange resin may be added to neutralize the treatment liquid.
【００３９】陰イオン交換樹脂とは、各種陽イオン性樹
脂のＯＨ形又は弱酸塩形のものであり、陽イオン性の樹
脂としては、強塩基性の陰イオン交換樹脂から弱塩基性
の陰イオン交換樹脂まで、あらゆるタイプの陰イオン交
換樹脂を包含する。また、これら陽イオン性樹脂の弱酸
塩形を構成する弱酸類としては、炭酸、蟻酸，酢酸など
の有機酸が好ましい。陽イオン性樹脂で特に好ましいも
のは、強塩基性のトリメチルアミノ基（Ｉ型）やヒドロ
キシエチルジメチルアミノ基（ＩＩ型）を有する陰イオ
ン交換樹脂などが有る。陽イオン交換樹脂としては前記
のものがある。糖類を主とすると干渉物質の除去に多種
類の樹脂を必要とする組み合せにおいては、１本のカラ
ムに全ての樹脂が層状になる様重ねて充填するか、混合
して充填すれば良い。層状に重ねて充填する場合、糖類
を除去する樹脂を上層に、中和用樹脂を下層に充填する
のが好ましい。The anion exchange resin is an OH type or a weak acid salt type of various cationic resins. Examples of the cationic resin include a strong basic anion exchange resin and a weak basic anion. It includes all types of anion exchange resins, up to the exchange resin. Organic acids such as carbonic acid, formic acid and acetic acid are preferable as the weak acids constituting the weak acid salt form of these cationic resins. Particularly preferred cationic resins include anion exchange resins having a strongly basic trimethylamino group (type I) or a hydroxyethyldimethylamino group (type II). Examples of the cation exchange resin include those described above. In a combination that requires many kinds of resins for removing an interfering substance, mainly saccharides, one column may be filled with all the resins layered or mixed and filled. When filling in layers, it is preferable to fill the upper layer with the resin from which saccharides are removed and fill the lower layer with the neutralizing resin.
【００４０】第４の発明のを実施するには検体に、タ
ンパク質を油状沈澱させうるタンパク質不溶化剤を添加
し、タンパク質を沈澱させた後、その上清を上記のカラ
ムに通過させ、カラムを水洗後通過液中の糖アルコール
を定量すればよい。第４の発明のにおいて、タンパク
質の沈澱剤としては例えばトリクロル酢酸、過塩素酸、
硫酸、塩酸などの強酸類が有る。沈澱剤の添加量は０．
５〜１０％であるが、使用する検体及び変成剤の種類と
量によって変化し、血清にＳＤＳを添加した場合のトリ
クロル酢酸の至適使用量は２〜６％となる。又、タンパ
ク質変成剤とは沈澱剤により沈澱したタンパク質の再溶
解を防止するために加えるものであり、ドデシルベンゼ
ンスルホン酸ナトリウム（以下ＳＤＳと言う）、タング
ステン酸ナトリウム、モリブデン酸ナトリウム、ベンジ
ンアルコールなどが有り、中でもＳＤＳがより好まし
い。ＳＤＳを用いた場合の添加量は、検体中のタンパク
質を完全に変成させ得る十分な量であれば良く、血清の
場合０．２％〜５％であるが、より好ましくは０．５〜
２％である。In practicing the fourth invention, a protein insolubilizing agent capable of precipitating the protein in oil is added to the sample, and after the protein is precipitated, the supernatant is passed through the above column, and the column is washed with water. The sugar alcohol in the after-pass liquid may be determined. In the fourth invention, as a protein precipitant, for example, trichloroacetic acid, perchloric acid,
There are strong acids such as sulfuric acid and hydrochloric acid. The amount of the precipitant added is 0.
The amount is 5 to 10%, but varies depending on the type and amount of the specimen and the denaturant used, and the optimal use amount of trichloroacetic acid when SDS is added to serum is 2 to 6%. The protein denaturant is added to prevent the protein precipitated by the precipitant from re-dissolving, and includes sodium dodecylbenzenesulfonate (hereinafter referred to as SDS), sodium tungstate, sodium molybdate, benzene alcohol, and the like. Yes, especially SDS. The amount of addition in the case of using SDS may be an amount sufficient to completely denature the protein in the sample, and is 0.2% to 5% in the case of serum, and more preferably 0.5% to 5%.
2%.
【００４１】上記のタンパク質変成剤と沈澱剤はそれぞ
れ単独に２つの溶液として構成し、順次検体に添加する
が、場合によっては、それぞれ混合した溶液とし一度に
添加しても良い。又、カラムに充填する塩基性陰イオン
交換樹脂としては上記発明２で使用するものと同じも
のが使用され、又、中和剤として陽イオン交換樹脂も併
用しうる。The above-mentioned protein denaturing agent and precipitating agent are each independently constituted as two solutions, and are sequentially added to the sample. However, depending on the case, they may be added as a mixed solution at once. As the basic anion exchange resin to be packed in the column, the same one as used in the above-mentioned invention 2 is used, and a cation exchange resin may be used in combination as a neutralizing agent.
【００４２】この発明４のの方法を実施するには、検
体にタンパク質変性剤と沈澱剤を添加し、タンパク質を
水不溶性の沈澱とした後、沈澱を遠心分離することな
く、そのまま樹脂を充填したカラムに通し、水洗後、通
過液中の糖アルコールを定量すればよい。なお、ここで
使用するカラムは不溶化したタンパク質をろ過して除去
できるものであれば何でもよいが、濾紙、表面親水化処
理ポリエチレン樹脂の焼結シートなどのフィルターを樹
脂の上部に取りつけたものの方が好ましい。In order to carry out the method of the invention 4, a protein denaturing agent and a precipitant were added to the sample to make the protein a water-insoluble precipitate, and the resin was filled as it was without centrifuging the precipitate. After passing through a column and washing with water, the sugar alcohol in the passing solution may be quantified. The column used here may be of any type as long as it can remove insolubilized proteins by filtration.However, a column in which a filter such as filter paper or a sintered sheet of surface-hydrophilized polyethylene resin is attached to the upper part of the resin is better. preferable.
【００４３】第４の発明のを実施する場合の１検体当
りの使用量は例えば次のとおり、 タンパク質の変成沈澱剤 タンパク質変成剤：ＳＤＳ ２〜２０μｇ 沈 澱 剤 ：トリクロロ酢酸 ５〜３０μｇ カ ラ ム ろ過フィルター １枚以上 強塩基性陰イオン交換樹脂（ＯＨ形） ０．１〜１．０ｍｌ 陽イオン交換樹脂 ０．０１〜０．５ｍｌ 全量で０．２〜１．５ｍｌ 又は ろ過フィルター １枚以上 強塩基性陰イオン交換樹脂（ホウ酸形） ０．１〜０．５ｍｌ 強塩基性陰イオン交換樹脂（ＯＨ形） ０．１〜１．０ｍｌ 陽イオン交換樹脂 ０．０１〜０．５ｍｌ 全量で０．５〜２ｍｌThe amount used per sample when carrying out the fourth invention is, for example, as follows: Protein denaturing and precipitating agent Protein denaturing agent: 2-20 μg of SDS Precipitating agent: 5-30 μg of trichloroacetic acid Filtration filter 1 or more Strongly basic anion exchange resin (OH form) 0.1-1.0 ml Cation exchange resin 0.01-0.5 ml 0.2-1.5 ml in total volume or more than 1 filtration filter Basic anion exchange resin (borate form) 0.1-0.5 ml Strongly basic anion exchange resin (OH form) 0.1-1.0 ml Cation exchange resin 0.01-0.5 ml 0 in total volume 0.5-2 ml
【００４４】第４の発明のを実施する場合の１検体当
りの使用量は例えば次のとおり、 油状沈澱を生じさせるタンパク不溶化剤 アクリノール ２〜２０μｇ カ ラ ム ダイヤイオン ＨＰ−２０ ＳＳ ０．１〜０．５ｍｌ 強塩基性陰イオン交換樹脂（ＯＨ形） ０．１〜１．０ｍｌ 陽イオン交換樹脂 ０．０１〜０．５ｍｌ 全量で０．５〜２ｍｌ 又は ダイヤイオン ＨＰ−２０ ＳＳ ０．１〜０．５ｍｌ 強塩基性陰イオン交換樹脂（ホウ酸形） ０．１〜０．５ｍｌ 強塩基性陰イオン交換樹脂（ＯＨ形） ０．１〜１．０ｍｌ 陽イオン交換樹脂 ０．０１〜０．５ｍｌ 全量で０．５〜２．０ｍｌThe amount used per sample when carrying out the fourth invention is, for example, as follows: 2 to 20 μg of a protein insolubilizer Acrynol which causes an oily precipitate; Caram Diaion HP-20 SS 0.1 to 0.1; 0.5 ml Strongly basic anion exchange resin (OH form) 0.1-1.0 ml Cation exchange resin 0.01-0.5 ml 0.5-2 ml in total volume or Diaion HP-20 SS 0.1- 0.5 ml Strongly basic anion exchange resin (borate form) 0.1-0.5 ml Strongly basic anion exchange resin (OH form) 0.1-1.0 ml Cation exchange resin 0.01-0. 5ml 0.5 ~ 2.0ml in total volume
【００４５】[0045]
【実施例】次に、実験例、実施例により、本発明を具体
的に説明する。Next, the present invention will be described specifically with reference to experimental examples and examples.
【００４６】実験例１． （ピラノースオキシダーゼを使用した。ＡＧの検量線）
０．２５Ｍのリン酸緩衝液（ｐＨ５．６）にピラノース
オキシダーゼ（グルコースに対する比活性５単位／ｍ
ｇ、宝酒造製）２ｍｇ／ｍｌ、ＨＲＰ０．２４単位／ｍ
ｌ、ＡＢＴＳ ４ｍＭとなる様に溶解しＡＧ検出試薬を
作成した。ＡＧ標準液０．１ｍｌに蒸留水２ｍｌ添加し
てＡＧ溶液とし、さらに前記のＡＧ検出試薬０．５ｍｌ
を加えて２５℃で１時間反応した。この反応液の４２０
ｎｍにおける吸光度を測定して検量線を作成したものを
図１に示した。Experimental Example 1 (Pyranose oxidase was used. Standard curve of AG)
Pyranose oxidase (specific activity for glucose 5 units / m2) was added to a 0.25 M phosphate buffer (pH 5.6).
g, Takara Shuzo) 2 mg / ml, HRP 0.24 unit / m
1, ABTS was dissolved to 4 mM to prepare an AG detection reagent. 2 ml of distilled water was added to 0.1 ml of the AG standard solution to prepare an AG solution, and further, 0.5 ml of the AG detection reagent described above.
Was added and reacted at 25 ° C. for 1 hour. 420 of this reaction solution
FIG. 1 shows a calibration curve prepared by measuring the absorbance at nm.
【００４７】実験例２． （Ｌ−ソルボースオキシダーゼを使用したＡＧの検量
線）実験例１のピラノースオキシダーゼの代りにＬ−ソ
ルボースオキシダーゼ（グルコースに対する比活性４．
３単位／ｍｇに変えて全く同様に反応させ、ＡＧの検量
線を作成した。その結果を図２に示した。Experimental Example 2 (Calibration curve of AG using L-sorbose oxidase) Instead of pyranose oxidase in Experimental Example 1, L-sorbose oxidase (specific activity to glucose 4.
The reaction was carried out in exactly the same manner except that the amount was changed to 3 units / mg, and a calibration curve for AG was prepared. The result is shown in FIG.
【００４８】実験例３． （ミオイノシトールデヒドロゲナーゼを使用したＭＩの
検量線）７０ｍＭ硫安を含む２０ｍＭ炭酸ナトリウム緩
衝液（ｐＨ９．０）で、ミオイノシトールデヒドロゲナ
ーゼ（１０単位／ｍｇ、シグマ社製）及びＮＡＤをそれ
ぞれ１単位／ｍｌ、１ｍＭとなる様に溶解しＭＩ検出試
薬を作成した。ＭＩ標準液０．１ｍｌに、ＭＩ検出試薬
０．９ｍｌを加え２５℃で２時間反応した。この反応液
のケイ光強度を励起波長３６５ｎｍ、検出波長４５０ｎ
ｍで測定し、その結果を図３に示した。Experimental Example 3 (Calibration curve of MI using myo-inositol dehydrogenase) In 20 mM sodium carbonate buffer (pH 9.0) containing 70 mM ammonium sulfate, myo-inositol dehydrogenase (10 units / mg, manufactured by Sigma) and NAD were each 1 unit / ml, It was dissolved to 1 mM to prepare an MI detection reagent. 0.9 ml of the MI detection reagent was added to 0.1 ml of the MI standard solution, and reacted at 25 ° C. for 2 hours. The fluorescence intensity of this reaction solution was measured at an excitation wavelength of 365 nm and a detection wavelength of 450 n.
m, and the results are shown in FIG.
【００４９】実施例１−１． （ピラノースオキシダーゼを使用するＡＧの測定）血清
検体を、後述した実施例１−４〜１−６に示す前処理を
ほどこして糖類を除去し、残存するＡＧの実験例１に示
した方法で測定した。ＡＧの定量は、それぞれのＡＧ標
準液を血清検体と全く同様の方法で前処理して作成した
検量線によって行った。その結果を表１に示した。Embodiment 1-1. (Measurement of AG Using Pyranose Oxidase) A serum sample was subjected to the pretreatment described in Examples 1-4 to 1-6 described below to remove saccharides, and the remaining AG was measured by the method described in Experimental Example 1 of AG. did. AG was quantified by a calibration curve prepared by pretreating each AG standard solution in exactly the same manner as the serum sample. The results are shown in Table 1.
【００５０】実施例１−２ （Ｌ−ソルボースオキシダーゼを使用するＡＧの測定）
実施例１−１で使用したものと同一の血清検体を後述し
た実施例１−４〜１−６に示した前処理をほどこして糖
類を除去し、残存するＡＧを実験例２に示した方法で測
定した。ＡＧの定量は、それぞれのＡＧ標準液を血清検
体と全く同様の方法で前処理して作成した検量線によっ
て行った。その結果を表１に示した。Example 1-2 (Measurement of AG using L-sorbose oxidase)
The same serum sample as used in Example 1-1 was subjected to the pretreatment described in Examples 1-4 to 1-6 described below to remove saccharides, and the remaining AG was subjected to the method described in Experimental Example 2. Was measured. AG was quantified by a calibration curve prepared by pretreating each AG standard solution in exactly the same manner as the serum sample. The results are shown in Table 1.
【００５１】[0051]
【表１】 [Table 1]
【００５２】実施例１−３． （ミオイノシトールデヒドロゲナアーゼを使用するＭＩ
の測定）尿検体を、後述した実施例１−７に示す前処理
をほどこして糖類を除去し、残存するＭＩを実験例３に
示した方法で測定した。ＭＩの定量は、それぞれＭＩ標
準液を尿検体と全く同様の方法で前処理して作成した検
量線によって行った。結果は同一尿検体をガスクロで測
定した値と良く一致した。Embodiment 1-3. (MI using myo-inositol dehydrogenase)
Measurement) The urine sample was subjected to the pretreatment described in Example 1-7 described below to remove saccharides, and the remaining MI was measured by the method described in Experimental Example 3. The quantification of MI was performed by a calibration curve prepared by pretreating the MI standard solution in exactly the same manner as the urine sample. The results agreed well with the values measured by gas chromatography on the same urine sample.
【００５３】実施例１−４． （血清検体の、タンパク質吸着樹脂を添加した干渉物質
除去カラムによる糖アルコール分析用前処理）イオン交
換樹脂ＡＧ５０Ｗ−Ｘ８Ｈ形、ＱＡＥ−トヨパール５５
０Ｃ（東ソ製）ホウ酸形をそれぞれ０．１ｍｌ、０．５
ｍｌづつ小カラムに下部より順次層状に充填し試料前処
理カラムを作成した。このカラムにヒト血清０．１ｍｌ
を直接導通後蒸留水０．５ｍｌで４回洗浄して通過液
２．１ｍｌを得た。この通過液中に回収された糖アルコ
ールのうち、ＡＧを定量する場合の例を、実験例１と同
様にして行った。同一検体のガスクロ法による測定値と
の相関を図６に示した。上記カラムにおいて、ＱＡＥ−
トヨパール５５０Ｃホウ酸形をそのＯＨに変え、他は上
記と同様にすると、本実施例と同様の結果を得た。Embodiment 1-4. (Pretreatment of serum sample for sugar alcohol analysis using an interference substance removal column to which protein adsorption resin is added) Ion exchange resin AG50W-X8H type, QAE-Toyopearl 55
0C (manufactured by Toso) boric acid form 0.1 ml, 0.5 ml
Each small column was packed in layers from the bottom in small columns to prepare a sample pretreatment column. 0.1 ml of human serum is added to this column.
Was directly passed through and washed four times with 0.5 ml of distilled water to obtain 2.1 ml of a passing solution. An example in which AG was quantified among the sugar alcohols recovered in the passing solution was performed in the same manner as in Experimental Example 1. FIG. 6 shows the correlation between the same sample and the value measured by the gas chromatography method. In the above column, QAE-
When the Toyopearl 550C boric acid form was changed to its OH, and the other conditions were the same as above, the same results as in this example were obtained.
【００５４】また、上記前処理カラムに充填する樹脂
を、ＡＧ５０Ｗ−Ｘ８ Ｈ形とＱＡＥ−トヨパール ホ
ウ酸形の１：５の混合樹脂に変え、他は上記と同様とす
ると、本実施例と同様の結果が得られた。なお、本実施
例で使用したＱＡＥ−Toyopearl のＯＨ形及びホウ酸形
の樹脂は市販のＣｌ形より次の方法で調製される。即
ち、ＱＡＥ−Toyopearl ５５０Ｃ（Ｃｌ形）２５０ｍ
ｌをカラムに充填し、０．２Ｍ水酸化ナトリウム溶液１
ｌをゆっくり流してＣｌイオンを流出させた後、蒸留水
で洗浄し、カラム流出廃液が中性になるので洗浄し、Ｏ
Ｈ形の樹脂を得る。このＯＨ形の樹脂に、０．５Ｍホウ
酸溶液３ｌを上部よりゆっくり流し、流出廃液のｐＨが
酸性になるまで中和する。ついで蒸留水で流出廃液が中
性になるまで洗浄し、ホウ酸形の樹脂を得る。又、ＡＧ
５０Ｗ−Ｘ８のＨ形は市販のＮａ形より次の方法で調製
される。即ち、ＡＧ５０Ｗ−Ｘ８（Ｎａ形）２００ｍｌ
をカラムに充填し、ＩＭ塩酸１リットルをゆっくり流し
てＮａイオンを流出させた後、蒸留水で洗浄し、流水廃
液のｐＨを中性とする。Also, the resin to be packed in the pretreatment column was changed to a mixed resin of 1:50 of AG50W-X8 H form and QAE-Toyopearl boric acid form, and the other conditions were the same as in the above example. Was obtained. The OH type and boric acid type resins of QAE-Toyopearl used in this example are prepared from commercially available Cl type by the following method. That is, QAE-Toyopearl 550C (Cl type) 250 m
to a column, 0.2 M sodium hydroxide solution 1
1 to slowly elute Cl ions, and then wash with distilled water.
An H-shaped resin is obtained. 3 L of a 0.5 M boric acid solution is slowly flowed from above to the OH-type resin, and neutralized until the pH of the waste liquid is acidic. Subsequently, the effluent is washed with distilled water until the waste liquid becomes neutral, thereby obtaining a boric acid type resin. AG
Form H of 50W-X8 is prepared from commercially available Na form in the following manner. That is, AG50W-X8 (Na type) 200 ml
Is packed in a column, and 1 liter of IM hydrochloric acid is slowly flowed to elute Na ions. Then, the column is washed with distilled water to neutralize the pH of the wastewater of running water.
【００５５】実施例１−５． （血清検体の不可逆的タンパク不溶化剤とろ過フィルタ
ー付干渉物質除去カラムによる糖アルコール分析用前処
理）イオン交換樹脂ＡＧ５０Ｗ−Ｘ８ Ｈ形、ＡＧ１−
Ｘ８ ＯＨ形（いづれもバイオラッド社製）をそれぞれ
０．１ｍｌ、０．４ｍｌづつ小カラムに下部より順次層
状に充填し、充填した樹脂の最上部に親水性ポリエチレ
ンの焼結フィルターをセットし、試料前処理カラムを作
製した。ヒト血清０．２ｍｌ〜５％ＳＤＳ溶液０．１ｍ
ｌを加えて振トウの後、さらに１２％トリクロル酢酸水
溶液０．１ｍｌを加えて強く振トウした。得られた沈澱
物を含む分散液の０．２ｍｌを上記の試料前処理カラム
導通し、蒸留水０．５ｍｌで４回洗浄して通過液２．２
ｍｌを得た。この通過液中に回収された糖アルコールの
うち、ＡＧを定量する場合の例を以下に示す。ＡＧの測
定は、実験例１に示した方法で行った。即ち、このＡＧ
溶液にＡＧ検出試薬０．５ｍｌを直接添加して反応させ
た。さらに、ＡＧの標準液を用いて作成した検量線を用
いて定量した。この様にして測定した正常人及び糖尿病
患者の血清ＡＧ値と、同一検体をガスクロ法で測定した
場合の血清ＡＧ値の相関を、図４に示した。この図から
明らかなように、本発明の方法による定量値は、ガスク
ロ法による定量値と高い相関性を示し、大過剰に存在す
るグルコースの影響を全く受けなかった。Embodiment 1-5. (Pretreatment for the analysis of sugar alcohols using an irreversible protein insolubilizing agent for serum samples and an interference substance removal column with a filtration filter) Ion exchange resin AG50W-X8 H type, AG1-
X8OH form (both manufactured by Bio-Rad Co., Ltd.) is packed in small columns in order of 0.1 ml and 0.4 ml respectively from the bottom in layers, and a sintered filter of hydrophilic polyethylene is set on top of the filled resin. A sample pretreatment column was prepared. Human serum 0.2ml-5% SDS solution 0.1m
After the addition of 1 l, the mixture was shaken tough, and 0.1 ml of a 12% aqueous solution of trichloroacetic acid was further added to shake vigorously. 0.2 ml of the resulting dispersion containing the precipitate was passed through the sample pretreatment column, washed four times with 0.5 ml of distilled water, and passed through 2.2.
ml were obtained. An example in which AG is quantified among the sugar alcohols recovered in the passing solution is described below. AG was measured by the method shown in Experimental Example 1. That is, this AG
0.5 ml of an AG detection reagent was directly added to the solution to cause a reaction. Furthermore, quantification was performed using a calibration curve prepared using an AG standard solution. FIG. 4 shows the correlation between the serum AG values of the normal person and the diabetic patient thus measured and the serum AG values when the same sample was measured by the gas chromatography method. As is clear from this figure, the quantitative value obtained by the method of the present invention showed a high correlation with the quantitative value obtained by the gas chromatography method, and was not affected by the glucose present in a large excess.
【００５６】実施例１−６． （血清検体の、油状沈澱を生じさせるタンパク不溶化剤
と不溶化剤吸着樹脂添加干渉物質除去カラムによる糖ア
ルコール分析用前処理）イオン交換樹脂ＡＧ５０Ｗ−Ｘ
８ Ｈ形、ＡＧ１−Ｘ８ ０Ｈ形（いずれもバイオラッ
ド社製）、疎水性樹脂ダイヤイオンＨＰ−２０ＳＳ（三
菱化成製）をそれぞれ０．１ｍｌ、０．３ｍｌ、０．１
ｍｌづつ小コラムに下部より順次層状に充填し試料前処
理カラムを作製した。ヒト血清０．２ｍｌへ２．５％ア
クリノール０．２ｍｌを加えて振トウした。血清中のタ
ンパク質は油状の沈澱となって沈降するので、この上清
部分の０．２ｍｌを上記の飼料前処理カラムへ導通し、
蒸留水０．５ｍｌで４回洗浄して通過液２．２ｍｌを得
た。この通過液中に回収された糖アルコールのうち、Ａ
Ｇを定量とする場合の例を、実施例１−４と同様にして
行った。同一検体のガスクロ法による測定値との相関を
図５に示した。Embodiment 1-6. (Pretreatment for analysis of sugar alcohol by serum interference sample removing column with addition of protein insolubilizing agent and insolubilizing agent-adsorbing resin causing oily precipitation) Ion exchange resin AG50W-X
8H type, AG1-X80H type (all manufactured by Bio-Rad) and hydrophobic resin Diaion HP-20SS (Mitsubishi Kasei) were 0.1 ml, 0.3 ml, 0.1 ml, respectively.
Each small column was filled in layers sequentially from the bottom into small columns to prepare a sample pretreatment column. 0.2 ml of 2.5% acrinol was added to 0.2 ml of human serum and shaken. Since the protein in the serum precipitates as an oily precipitate, 0.2 ml of the supernatant is passed through the above-mentioned feed pretreatment column,
After washing four times with 0.5 ml of distilled water, 2.2 ml of a passing solution was obtained. Among the sugar alcohols recovered in this passing liquid, A
An example in which G was determined was performed in the same manner as in Example 1-4. FIG. 5 shows the correlation between the same sample and the value measured by the gas chromatography method.
【００５７】実施例１−７． （尿検体のタンパク吸着樹脂を添加した干渉物質除去カ
ラムによる糖アルコール分析用前処理）イオン交換樹脂
ＡＧ５０Ｗ−Ｘ８ Ｈ形、ＱＡＥ−トヨパールホウ酸形
をそれぞれ０．２ｍｌ．１．８ｍｌづつ小カラムに下部
より順次層状に充填し、試料前処理カラムを作成した。
このカラムにヒト尿０．５ｍｌを直接導通後、蒸留水
１．０ｍｌで８回洗浄して通過液８．５ｍｌを得た。こ
の通過液中に回収された糖アルコールのうち、ＭＩを定
量する場合の例を以下に示す。上記の通過液８．５ｍｌ
全量を濃縮乾固し、蒸留水０．１ｍｌで再溶解して前処
理カラムの処理液を得た。ＭＩの検出は実験例３に従っ
て行った。即ち、この処理液に、直接ＭＩ検出試薬を
０．９ｍｌ添加して反応させた。さらに、ＭＩの定量は
ＭＩ標準液を用いて作成した検量線を用いて行った。こ
の様にして測定した正常人と糖尿病患者の尿中ＭＩ値
を、同一検体のガスクロ法での測定値と比較した結果を
表２に示した。Embodiment 1-7. (Pretreatment for analysis of sugar alcohol by means of an interference substance removal column to which a protein adsorption resin of a urine sample was added) 0.2 ml of each of ion-exchange resins AG50W-X8 H form and QAE-Toyopearl borate form. 1.8 ml each of the small columns were sequentially packed in layers from the bottom to form a sample pretreatment column.
After directly passing 0.5 ml of human urine through the column, the column was washed eight times with 1.0 ml of distilled water to obtain 8.5 ml of a permeate. An example in which MI is quantified among the sugar alcohols recovered in the passing liquid is described below. 8.5 ml of the above passing liquid
The whole amount was concentrated to dryness and redissolved in 0.1 ml of distilled water to obtain a treatment liquid for a pretreatment column. The detection of MI was performed according to Experimental Example 3. That is, 0.9 ml of the MI detection reagent was directly added to the treatment liquid to cause a reaction. Furthermore, the quantification of MI was performed using a calibration curve prepared using the MI standard solution. Table 2 shows the results of comparing the urinary MI values of the normal person and the diabetic patient thus measured with the values measured by the gas chromatography method of the same sample.
【００５８】[0058]
【表２】 [Table 2]
【００５９】実施例１−８． （血清ＡＧ測定キットの例） （１）キットの調整 ＡＧ検出試薬：ＰＲＯＤ２００ｍｇ（５単位／ｍｇ、宝
酒造製）、ＨＲＰ０．２４ｍｇ（１００Ｕ／ｍｇ、和光
純薬製）及びＡＢＴＳ２２０ｍｇ（ベーリンガー社製）
を０．２５Ｍリン酸ナトリウム緩衝液（ｐＨ５．６）１
００ｍｌを溶解し、１６ｍｌづつバイアルに分注し、常
法により凍結乾燥した。 復元液：トリトン；Ｘ−４０５（和光純薬製）０．６ｇ
を蒸留水１００ｍｌに溶解し、バイアルに１６ｍｌづつ
分注して栓をした。 標準液：５ｍｇのＡＧを１００ｍｌの蒸留水で溶解し、
バイアルに３ｍｌづつ分注して栓をした。 前処理カラム：フリットフィルターを装着したリザーバ
ー（１．５ｍｌ容量、アナリティカルインターナショナ
ル社製）に、下から順にＡＧ５０Ｗ−Ｘ８ Ｈ形（バイ
オラッド社製）０．１ｍｌとＱＡＥ−トヨパールホウ酸
形（東ソ社製）０．５ｍｌを充填した。さらに充填した
樹脂上からフリットフィルターを装着し、樹脂が動かな
い様に固定した。また、充填した樹脂の安定性を保ちか
つ乾燥しない様に０．２Ｍのホウ酸溶液をカラム上部に
満たし、出口にはキヤップを取り付け入口にはシールを
して密封した。Embodiment 1-8. (Example of serum AG measurement kit) (1) Preparation of kit AG detection reagent: PROD 200 mg (5 units / mg, manufactured by Takara Shuzo), HRP 0.24 mg (100 U / mg, manufactured by Wako Pure Chemical) and ABTS 220 mg (manufactured by Boehringer)
With 0.25 M sodium phosphate buffer (pH 5.6) 1
After dissolving 00 ml, the mixture was dispensed into vials 16 ml at a time and freeze-dried by a conventional method. Restoration liquid: Triton; 0.6 g of X-405 (manufactured by Wako Pure Chemical Industries)
Was dissolved in 100 ml of distilled water, and the solution was dispensed into vials in 16 ml portions and stoppered. Standard solution: 5 mg of AG is dissolved in 100 ml of distilled water,
The vial was dispensed in 3 ml portions and stoppered. Pretreatment column: 0.1 ml of AG50W-X8 H form (manufactured by Bio-Rad) and QAE-Toyopearl boric acid form (Tososo) in a reservoir (1.5 ml capacity, manufactured by Analytical International) equipped with a frit filter in order from the bottom. 0.5 ml). Further, a frit filter was attached from above the filled resin, and fixed so that the resin did not move. A 0.2 M boric acid solution was filled in the upper portion of the column so as to keep the stability of the filled resin and prevent drying, and a cap was provided at the outlet, and the inlet was sealed with a seal.
【００６０】（２）操作法 前処理カラムのキャップとシールを取り除きリザーバー
内のホウ酸溶液を排液し、さらに１ｍｌの蒸留水で２回
洗浄することにより、樹脂内の過剰のホウ酸を完全に洗
い流した。この洗浄した前処理カラムを試験管の上にセ
ットし、血清検体１００μｌを直接導通後、さらに蒸留
水０．５ｍｌで４回洗浄して通過液２．１ｍｌを得た。
ＡＧ検出試薬１バイアルを復元液１バイアルで復元し、
この溶液０．５ｍｌを、上記の前処理カラム通過液２．
１ｍｌに加えて攪拌し、２５℃で１時間反応した。この
反応液の４２０ｎｍにおける吸光度を通常の分光光度計
により測定した。また標準液を蒸留水で倍々希釈して希
釈系列を作成し、これも血清検体の場合と同様に処理し
て反応させ、ＡＧの検量線を作成した。血清検体中のＡ
Ｇ濃度は、この検量線を用い検体の吸光度値から計算し
た。キットの方法によっても、実施例１−４と同様の結
果が得られた。(2) Operating method The cap and seal of the pretreatment column are removed, the boric acid solution in the reservoir is drained, and the resultant is washed twice with 1 ml of distilled water to completely remove excess boric acid in the resin. Washed off. The washed pretreated column was set on a test tube, and 100 μl of a serum sample was directly passed through the column, followed by washing four times with 0.5 ml of distilled water to obtain 2.1 ml of a passing solution.
Reconstitute one vial of AG detection reagent with one vial of reconstitution solution,
0.5 ml of this solution was added to the solution passed through the pretreatment column described above.
The mixture was added to 1 ml, stirred, and reacted at 25 ° C. for 1 hour. The absorbance at 420 nm of this reaction solution was measured with a usual spectrophotometer. Further, the standard solution was diluted twice with distilled water to prepare a dilution series, which was processed and reacted in the same manner as in the case of the serum sample, and an AG calibration curve was prepared. A in serum sample
The G concentration was calculated from the absorbance value of the sample using this calibration curve. The same results as in Examples 1-4 were obtained by the kit method.
【００６１】実施例２−１ 測定は、ポンプから、０．０５Ｍ−ほう酸緩衝液（ｐＨ
５．８）を０．５ｍｌ／ｍｉｎの流速で送液し、オート
インジェクター、干渉物質除去カラム、バイオセンサー
とこれに連結したデータ処理機からなる装置で行った。
バイオセンサーは、フローセル（容量約１００μｌにつ
いた過酸化水素電極（（株）エイブル社製）の表面（５
ｍｍφ）に同じ大きさのＰＲＯＤ固定膜をナイロンネッ
トで装着して使用した。ＰＲＯＤ固定化膜は、次の方法
により作成した。ＰＲＯＤ（宝酒造（株）製５．４Ｕ／
ｍｇ）１０ｍｇと牛血清アルブミン（シグマ社製）６ｍ
ｇを１／１５Ｍ−リン酸緩衝液（ｐＨ７．２）０．６ｍ
ｌに溶解し、１％グルタルアルデヒド水溶液０．２ｍｌ
を加え混合する。混合後直ちにニトロセルロース膜（２
５ｍｍφ×孔径３μｍ）２枚の上にゆっくり滴下し、全
体が均一になるように広げて、４℃で一夜風乾して得
た。Example 2-1 The measurement was carried out by using a 0.05M borate buffer (pH
5.8) was sent at a flow rate of 0.5 ml / min, and was carried out using a device comprising an auto injector, a column for removing interfering substances, a biosensor and a data processor connected to the biosensor.
The biosensor has a surface (5) of a flow cell (a hydrogen peroxide electrode (manufactured by Able Co., Ltd.) with a volume of about 100 μl).
mmφ), a PROD immobilization membrane of the same size was used with a nylon net. The PROD-immobilized membrane was prepared by the following method. PROD (Takara Shuzo 5.4U /
mg) 10 mg and bovine serum albumin (Sigma) 6 m
g of 0.65 m in 1/15 M phosphate buffer (pH 7.2)
1% glutaraldehyde aqueous solution 0.2ml
And mix. Immediately after mixing, the nitrocellulose membrane (2
(5 mmφ × pore size 3 μm) The solution was slowly dropped on two sheets, spread so as to be uniform, and air-dried at 4 ° C. overnight.
【００６２】干渉物質除去カラムは、５ｍｍφ×１００
ｍｍのカラムにポリビニルアルコール系のＱＡＥ−トヨ
パール５５０Ｃ（東ソ−（株）社製）を通常の方法でＯ
Ｈ形としさらにほう酸形とした樹脂と強カチオン交換樹
脂ＡＧ５０Ｗ−Ｘ８（Ｈ形）（バイオラッド社製）を
５：１に混合した樹脂を充填したカラムを用いた。この
装置で、オートインジェクター（２０μｌ固定ループ）
にＡＧの標準１，１０，４０μｇ／ｍｌを並べ、次いで
血清サンプル１０本並べて、測定を開始した。データ処
理機により、ＡＧの標準の面積値から検量線を作製し、
サンプルのＡＧ定量値が得られた。また、同一検体をＧ
Ｃ法で測定した定量値と相関を図７に示した。この図か
ら明らかなように、本発明による定量値は、ＧＣ法と高
い相関を示した。The interference substance removal column is 5 mmφ × 100
mm column of polyvinyl alcohol-based QAE-Toyopearl 550C (manufactured by Tosoh Corporation) in a usual
A column packed with a resin obtained by mixing the resin in the H form and the boric acid form with a strong cation exchange resin AG50W-X8 (H form) (manufactured by Bio-Rad) at a ratio of 5: 1 was used. With this device, an auto-injector (20 μl fixed loop)
Then, 1, 10, 40 μg / ml of the AG standard was arranged, and then 10 serum samples were arranged to start measurement. Using a data processor, a calibration curve was created from the standard area values of AG,
The AG quantitative value of the sample was obtained. In addition, the same sample
FIG. 7 shows the quantitative values and correlations measured by the method C. As is clear from this figure, the quantitative value according to the present invention showed a high correlation with the GC method.
【００６３】実施例２−２ 測定装置は、ポンプ２台、オートインジェクター、干渉
物質除去カラム、バイオセンサーとこれに連結したデー
タ処理機からなっており、オートインジェクターに接続
して干渉物質除去カラムに水０．５ｍｌ／ｍｉｎの流速
で流し、カラム通過液は、０．１ｍｌ／ｍｉｎの流速で
供給される０．５Ｍ−リン酸緩衝液とミキシングジョイ
ントを通じて混合され、ミキシングコイル（１．０ｍｍ
φ×３ｍ）にて完全に混合された実施例２−１と同じバ
イオセンサーのフローセルに入るように接続された装置
である。干渉物質除去カラムは、５ｍｍφ×１００ｍｍ
のカラムにＱＡＥ−トヨパール５５０Ｃ（ほう酸形）を
充填したものを用いた。この装置で、実施例２−１と同
様に測定した。また、同一検体をＧＣ法で測定した定量
値との相関を図８に示した。この図から明らかなよう
に、本発明による定量値は、ＧＣ法と高い相関を示し
た。Example 2-2 The measuring device was composed of two pumps, an auto-injector, an interfering substance removing column, a biosensor and a data processor connected thereto, and was connected to the auto-injector to provide an interfering substance removing column. Water is flowed at a flow rate of 0.5 ml / min, and the liquid passing through the column is mixed with a 0.5 M phosphate buffer supplied at a flow rate of 0.1 ml / min through a mixing joint, and mixed with a mixing coil (1.0 mm
This is a device connected so as to enter the flow cell of the same biosensor as in Example 2-1 which was completely mixed at φ × 3 m). Interference substance removal column is 5mmφ × 100mm
The column packed with QAE-Toyopearl 550C (borate type) was used. With this apparatus, measurement was performed in the same manner as in Example 2-1. FIG. 8 shows the correlation between the same sample and a quantitative value measured by the GC method. As is clear from this figure, the quantitative value according to the present invention showed a high correlation with the GC method.
【００６４】実施例２−３ 測定装置は、実施例２−２と同様なシステムで行い、干
渉物質除去カラムに０．０１Ｍ−ほう酸緩衝液（ｐＨ
６．０）を１．０ｍｌ／ｍｉｎの流速で流し、もう一方
から１．１Ｍ−りん酸緩衝液（ｐＨ５．８）を０．１ｍ
ｌ／ｍｉｎの流速で供給して使用した。干渉物質除去カ
ラムは、５ｍｍφ×１００ｍｍのカラムにアクリル系の
シヨウデックスＴＭ−Ｌ（昭和電工（株）製）（ほう酸
形）を充填したものを用いた。この装置で、実施例２−
１と同様にして、血清サンプル２０本を測定した。ま
た、同一検体をＧＣ法で測定した定量値との相関を図９
に示した。この図から明らかなように、本発明による定
量値は、ＧＣ法と高い相関を示した。Example 2-3 The measurement was carried out using the same system as in Example 2-2, and a 0.01 M-borate buffer (pH
6.0) at a flow rate of 1.0 ml / min, and 1.1 M-phosphate buffer (pH 5.8)
It was supplied and used at a flow rate of 1 / min. As the column for removing the interfering substance, a column of 5 mmφ × 100 mm packed with acrylic-based SHIOdex TM-L (manufactured by Showa Denko KK) (borate type) was used. With this device, the embodiment 2
In the same manner as in Example 1, 20 serum samples were measured. FIG. 9 shows the correlation between the same sample and the quantitative value measured by the GC method.
It was shown to. As is clear from this figure, the quantitative value according to the present invention showed a high correlation with the GC method.
【００６５】[0065]
【発明の効果】以上から明らかなように、本発明の方法
によれば、検体中のタンパク質や糖類などの干渉物質が
簡単に除去でき、糖アルコール類の測定が極めて簡便に
なった。特定の糖アルコールに特異性は有るが、糖類と
幅広く反応する様な酵素であっても、本発明の糖類除去
の前処理法を組み合せれば、充分定量用に用い得ること
が明らかとなった。As is clear from the above, according to the method of the present invention, interference substances such as proteins and saccharides in a sample can be easily removed, and the measurement of sugar alcohols has become extremely simple. Although specific sugar alcohols have specificity, it has become clear that even enzymes that react widely with saccharides can be used for quantification sufficiently by combining the pretreatment method for saccharide removal of the present invention. .
【図１】 ピラノースオキシダーゼを使用したＡＧの検
量線FIG. 1. Calibration curve of AG using pyranose oxidase
【図２】 Ｌ−ソルボースオキシダーゼを使用したＡＧ
の検量線FIG. 2. AG using L-sorbose oxidase
Calibration curve
【図３】 ミオイノシトールデヒドロゲナーゼを用いた
ＭＩの検量線FIG. 3: Calibration curve of MI using myo-inositol dehydrogenase
【図４】 第４のの発明により処理された血清をピラ
ノースオキシダーゼを用いて測定した値と、ガスクロ法
の相関FIG. 4 shows a correlation between a value obtained by measuring the serum treated according to the fourth invention using pyranose oxidase and a gas chromatography method.
【図５】 第４のの発明により処理された血清をピラ
ノースオキシダーゼを用いて測定した値とガスクロ法の
相関FIG. 5 shows a correlation between a value obtained by measuring pyranose oxidase in serum treated according to the fourth invention and a gas chromatography method.
【図６】 第１の発明により処理された血清をピラノー
スオキシダーゼを用いて測定した値と、ガスクロ法の相
関FIG. 6 shows a correlation between a value obtained by measuring the serum treated according to the first invention using pyranose oxidase and a gas chromatography method.
【図７】 実施例２−１の測定システムで血清中のＡＧ
を測定したときのＧＣ法との相関FIG. 7: AG in serum using the measurement system of Example 2-1
Correlation with GC method when measuring
【図８】 実施例２−２の測定システムで血清中のＡＧ
を測定したときの相関FIG. 8: AG in serum by the measurement system of Example 2-2
Correlation when measuring
【図９】 実施例２−３の測定システムで血清中のＡＧ
を測定したときのＧＣ法との相関FIG. 9: AG in serum using the measurement system of Example 2-3.
Correlation with GC method when measuring
───────────────────────────────────────────────────── フロントページの続き (72)発明者 加藤 和夫 埼玉県浦和市辻８−21−15−203 (72)発明者 田島 茂 群馬県藤岡市藤岡675−11 (72)発明者 橋場 正 群馬県新田郡笠懸村阿左美804−12 (72)発明者 速水 宏 群馬県高崎市岩鼻町239 (72)発明者 竹澤 智子 群馬県藤岡市藤岡1906−５ (72)発明者 平山 正近 埼玉県大宮市奈良町136−51奈良町団地７ −203 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuo Kato 8-21-15-203 Tsuji, Urawa-shi, Saitama (72) Inventor Shigeru Tajima 675-11 Fujioka, Fujioka-shi, Gunma Prefecture (72) Inventor Tadashi Hashiba Gunma Prefecture 804-12 Asami, Kasake-mura, Nitta-gun (72) Inventor Hiroshi Hayami 239, Iwana-cho, Takasaki-shi, Gunma (72) Inventor Tomoko Takezawa 1906-5, Fujioka, Fujioka-shi, Gunma 1906-5 (72) Inventor Masachika Hirayama, Omiya-shi, Saitama 136-51 Nara-cho 7-203 Nara-cho housing complex
Claims (2)
る検体に、油状沈澱を生じさせるタンパク質不溶化剤
を添加し、タンパク質を油状沈澱物として沈降させて上
清液を得、次いでその上清液をタンパク不溶化剤吸着樹
脂及び塩基性陰イオン交換樹脂を充填したカラムに通す
か又は、タンパク質沈澱剤及びタンパク質変性剤を添
加し、タンパク質を水不溶化物として析出させ、得られ
た析出液を塩基性陰イオン交換樹脂を充填したカラムに
通し、次いで、得られた通過液中の糖アルコールを定量
することを特徴とする糖アルコールの定量方法。A sample containing sugar alcohols, proteins, and saccharides, to which a protein insolubilizing agent that causes an oily precipitate is added, and the protein is precipitated as an oily precipitate to obtain a supernatant. Pass through a column packed with a protein insolubilizer adsorption resin and a basic anion exchange resin, or add a protein precipitant and a protein denaturant to precipitate the protein as a water insolubilized substance, and then wash the resulting precipitate with a basic anion. A method for quantifying a sugar alcohol, comprising passing the solution through a column filled with an ion exchange resin, and then quantifying the sugar alcohol in the obtained passing solution.
定量するにあたり、通液している干渉物質除去カラムと
インジェクターとバイオセンサーを備えた装置に多数の
検体を順次注入することを特徴とする１，５−アンヒド
ログルシトールの定量法。2. A method for quantitatively determining 1,5-anhydroglucitol in blood, comprising sequentially injecting a large number of samples into an apparatus provided with an interfering substance removal column, an injector, and a biosensor. Characteristic method for quantifying 1,5-anhydroglucitol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18029897A JP3183500B2 (en) | 1989-04-10 | 1997-06-23 | Sugar alcohol determination method |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8796389 | 1989-04-10 | ||
| JP1-87963 | 1989-04-10 | ||
| JP10471289 | 1989-04-26 | ||
| JP1-104712 | 1989-04-26 | ||
| JP18029897A JP3183500B2 (en) | 1989-04-10 | 1997-06-23 | Sugar alcohol determination method |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2092342A Division JP2701176B2 (en) | 1989-04-10 | 1990-04-09 | Sugar alcohol quantification methods, columns and kits |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH1062402A true JPH1062402A (en) | 1998-03-06 |
| JP3183500B2 JP3183500B2 (en) | 2001-07-09 |
Family
ID=27305642
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18029897A Expired - Fee Related JP3183500B2 (en) | 1989-04-10 | 1997-06-23 | Sugar alcohol determination method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3183500B2 (en) |
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|---|---|---|---|---|
| WO2004078309A1 (en) * | 2003-03-05 | 2004-09-16 | Nihon University | Protein separation method using asynchronous coil planet centrifugal machine |
| US8753832B2 (en) | 2005-06-13 | 2014-06-17 | Nippon Kayaku Kabushiki Kaisha | Method of assaying 1,5 anhydroglucitol by using whole blood and measurement kit |
| US8465940B2 (en) | 2006-12-14 | 2013-06-18 | Nippon Kayaku Kabushiki Kaisha | Method for electrochemically measuring 1,5-anhydroglucitol in whole blood |
| WO2008072702A1 (en) * | 2006-12-14 | 2008-06-19 | Nippon Kayaku Kabushiki Kaisha | Method for measuring 1,5-anhydroglucitol in whole blood, and sensor chip and measurement kit to be used in the method |
| KR101470661B1 (en) * | 2006-12-14 | 2014-12-09 | 니폰 가야꾸 가부시끼가이샤 | Method for measuring 1, 5-anhydroglucitol in whole blood, and sensor chip and measurement kit to be used in the method |
| JPWO2008072702A1 (en) * | 2006-12-14 | 2010-04-02 | 日本化薬株式会社 | Method for measuring 1,5-anhydroglucitol in whole blood, sensor chip and measurement kit used therefor |
| JP5166284B2 (en) * | 2006-12-14 | 2013-03-21 | 日本化薬株式会社 | Method for measuring 1,5-anhydroglucitol in whole blood, sensor chip and measurement kit used therefor |
| JP2008249538A (en) * | 2007-03-30 | 2008-10-16 | Seiko Epson Corp | Detection element |
| JP5068825B2 (en) * | 2007-11-05 | 2012-11-07 | 日本化薬株式会社 | Biosensor |
| WO2009060849A1 (en) * | 2007-11-05 | 2009-05-14 | Nippon Kayaku Kabushiki Kaisha | Biosensor |
| US8951395B2 (en) | 2007-11-05 | 2015-02-10 | Nippon Kayaku Kabushiki Kaisha | Biosensor |
| US8883439B2 (en) | 2008-07-23 | 2014-11-11 | Nippon Kayaku Kabushiki Kaisha | Blood component measurement method utilizing hemolyzed whole blood, and kit for the method |
| JP5234675B2 (en) * | 2008-12-08 | 2013-07-10 | 日本化薬株式会社 | Biosensor for electrochemical measurement of 1,5-anhydroglucitol, measurement method and measurement kit using the same |
| US8658012B2 (en) | 2008-12-08 | 2014-02-25 | Nippon Kayaku Kabushiki Kaisha | Biosensor for electrochemical measurement of 1,5-anhydroglucitol, and measuring method and measuring kit using the same |
| WO2010067769A1 (en) * | 2008-12-08 | 2010-06-17 | 日本化薬株式会社 | Biosensor for electrochemical measurement of 1,5-anhydroglucitol, and measuring method and measuring kit using the same |
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| JP3183500B2 (en) | 2001-07-09 |
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