JPS63216500A - Analytic element for determination of enzymatic activity - Google Patents
Analytic element for determination of enzymatic activityInfo
- Publication number
- JPS63216500A JPS63216500A JP5166187A JP5166187A JPS63216500A JP S63216500 A JPS63216500 A JP S63216500A JP 5166187 A JP5166187 A JP 5166187A JP 5166187 A JP5166187 A JP 5166187A JP S63216500 A JPS63216500 A JP S63216500A
- Authority
- JP
- Japan
- Prior art keywords
- group
- layer
- analytical element
- represent
- alkyl
- 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.)
- Pending
Links
- 230000002255 enzymatic effect Effects 0.000 title 1
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 22
- 150000003931 anilides Chemical class 0.000 claims abstract description 18
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 15
- 239000007800 oxidant agent Substances 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 11
- -1 potassium ferricyanide Chemical compound 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 10
- 125000002768 hydroxyalkyl group Chemical group 0.000 claims abstract description 7
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 claims abstract description 6
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 claims abstract description 4
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 claims abstract description 4
- 125000003545 alkoxy group Chemical group 0.000 claims abstract description 3
- 238000011161 development Methods 0.000 claims description 11
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 125000004422 alkyl sulphonamide group Chemical group 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- 125000004421 aryl sulphonamide group Chemical group 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims description 2
- 239000000758 substrate Substances 0.000 abstract description 15
- 230000001590 oxidative effect Effects 0.000 abstract description 4
- 125000005422 alkyl sulfonamido group Chemical group 0.000 abstract 1
- 125000005421 aryl sulfonamido group Chemical group 0.000 abstract 1
- 229910052736 halogen Inorganic materials 0.000 abstract 1
- 150000002367 halogens Chemical class 0.000 abstract 1
- 239000010410 layer Substances 0.000 description 86
- 239000007788 liquid Substances 0.000 description 15
- 238000001514 detection method Methods 0.000 description 10
- 230000007480 spreading Effects 0.000 description 9
- 238000003892 spreading Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 108010010803 Gelatin Proteins 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 229920000159 gelatin Polymers 0.000 description 8
- 239000008273 gelatin Substances 0.000 description 8
- 235000019322 gelatine Nutrition 0.000 description 8
- 235000011852 gelatine desserts Nutrition 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000004744 fabric Substances 0.000 description 5
- ADVGKWPZRIDURE-UHFFFAOYSA-N N-Ac-2-Aminophenol Natural products CC(=O)NC1=CC=CC=C1O ADVGKWPZRIDURE-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 229920001477 hydrophilic polymer Polymers 0.000 description 4
- 239000004094 surface-active agent Substances 0.000 description 4
- ASHGTJPOSUFTGB-UHFFFAOYSA-N 3-methoxyphenol Chemical compound COC1=CC=CC(O)=C1 ASHGTJPOSUFTGB-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000012790 adhesive layer Substances 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- LHGVFZTZFXWLCP-UHFFFAOYSA-N guaiacol Chemical compound COC1=CC=CC=C1O LHGVFZTZFXWLCP-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- ISPYQTSUDJAMAB-UHFFFAOYSA-N 2-chlorophenol Chemical compound OC1=CC=CC=C1Cl ISPYQTSUDJAMAB-UHFFFAOYSA-N 0.000 description 2
- HORNXRXVQWOLPJ-UHFFFAOYSA-N 3-chlorophenol Chemical compound OC1=CC=CC(Cl)=C1 HORNXRXVQWOLPJ-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-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
- 101000856500 Bacillus subtilis subsp. natto Glutathione hydrolase proenzyme Proteins 0.000 description 2
- 108020004206 Gamma-glutamyltransferase Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- SKZKKFZAGNVIMN-UHFFFAOYSA-N Salicilamide Chemical compound NC(=O)C1=CC=CC=C1O SKZKKFZAGNVIMN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 235000010724 Wisteria floribunda Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000001448 anilines Chemical class 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 208000028659 discharge Diseases 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- QLNWXBAGRTUKKI-UHFFFAOYSA-N metacetamol Chemical compound CC(=O)NC1=CC=CC(O)=C1 QLNWXBAGRTUKKI-UHFFFAOYSA-N 0.000 description 2
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- YXSKGOCVSTWEJU-UHFFFAOYSA-N n-(3-hydroxyphenyl)propanamide Chemical compound CCC(=O)NC1=CC=CC(O)=C1 YXSKGOCVSTWEJU-UHFFFAOYSA-N 0.000 description 2
- 239000000123 paper Substances 0.000 description 2
- 238000005375 photometry Methods 0.000 description 2
- 229920006267 polyester film Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000002759 woven fabric Substances 0.000 description 2
- ZTXWIKHKNGFJAX-UHFFFAOYSA-N 1-hydroxynaphthalene-2-carboxamide Chemical compound C1=CC=CC2=C(O)C(C(=O)N)=CC=C21 ZTXWIKHKNGFJAX-UHFFFAOYSA-N 0.000 description 1
- NGLHXBUDCGSGPT-UHFFFAOYSA-N 2-chloro-3-oxo-n,3-diphenylpropanamide Chemical compound C=1C=CC=CC=1C(=O)C(Cl)C(=O)NC1=CC=CC=C1 NGLHXBUDCGSGPT-UHFFFAOYSA-N 0.000 description 1
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108010008488 Glycylglycine Proteins 0.000 description 1
- 239000006173 Good's buffer Substances 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 150000003869 acetamides Chemical class 0.000 description 1
- HVYLDJKDVOOTHV-UHFFFAOYSA-N acetic acid;2-iminoethanethiol Chemical compound CC(O)=O.CC(O)=O.SCC=N HVYLDJKDVOOTHV-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 150000001639 boron compounds Chemical class 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001951 carbamoylamino group Chemical group C(N)(=O)N* 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 125000002642 gamma-glutamyl group Chemical group 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 229940043257 glycylglycine Drugs 0.000 description 1
- 229960001867 guaiacol Drugs 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical class [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229940100630 metacresol Drugs 0.000 description 1
- HNQIVZYLYMDVSB-UHFFFAOYSA-N methanesulfonimidic acid Chemical group CS(N)(=O)=O HNQIVZYLYMDVSB-UHFFFAOYSA-N 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 150000004780 naphthols Chemical class 0.000 description 1
- 239000004745 nonwoven fabric Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- JLKDVMWYMMLWTI-UHFFFAOYSA-M potassium iodate Chemical compound [K+].[O-]I(=O)=O JLKDVMWYMMLWTI-UHFFFAOYSA-M 0.000 description 1
- 239000001230 potassium iodate Substances 0.000 description 1
- 235000006666 potassium iodate Nutrition 0.000 description 1
- 229940093930 potassium iodate Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- JEXVQSWXXUJEMA-UHFFFAOYSA-N pyrazol-3-one Chemical class O=C1C=CN=N1 JEXVQSWXXUJEMA-UHFFFAOYSA-N 0.000 description 1
- 239000012070 reactive reagent Substances 0.000 description 1
- 229960000581 salicylamide Drugs 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid group Chemical class S(N)(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- BUUPQKDIAURBJP-UHFFFAOYSA-N sulfinic acid Chemical compound OS=O BUUPQKDIAURBJP-UHFFFAOYSA-N 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 229910000349 titanium oxysulfate Inorganic materials 0.000 description 1
- DCXPBOFGQPCWJY-UHFFFAOYSA-N trisodium;iron(3+);hexacyanide Chemical compound [Na+].[Na+].[Na+].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCXPBOFGQPCWJY-UHFFFAOYSA-N 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔発明の分野〕
本発明は分析素子に関し特にガンマグルタミルトランス
ペプチダーゼ（以下ｒ−ＧＴＰという）の活性測定に有
用な分析要素に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to an analytical element, and particularly to an analytical element useful for measuring the activity of gamma glutamyl transpeptidase (hereinafter referred to as r-GTP).
ｒ−ＧＴＰは、ある種の肝疾患において血液中に増大が
認められる酵素で、適当な酵素基質を用いて血中の酵素
活性を測定することにより、臨床上有意義な診断に対す
る知見を得ることができる。r-GTP is an enzyme that is found to increase in the blood in certain liver diseases, and by measuring the enzyme activity in the blood using an appropriate enzyme substrate, clinically meaningful diagnostic findings can be obtained. can.
従来、γ−し一グルタミルーバラニトロアニリドがこの
目的のための基質として使用されていたが、一般にこの
化合物は酵素反応を行なうためのｐＨでは極めて難溶で
あり、安定した測定値を得るための濃度に溶解させるた
めには界面活性剤を加えるなどの特別な操作を必要とし
た。Conventionally, γ-glutamyl-balanitroanilide has been used as a substrate for this purpose, but this compound is generally extremely sparingly soluble at the pH required for enzymatic reactions, and it is difficult to obtain stable measurement values. Special operations such as adding a surfactant were required to dissolve the compound at a concentration of .
特公昭５６−３５６６１号には溶解性のよいＴ−ＧＴＰ
基質として下記一般式で示される化合物が開示されてい
る。Special Publication No. 56-35661 contains T-GTP, which has good solubility.
A compound represented by the following general formula is disclosed as a substrate.
Ｃ）Ｉ！
■
Ｈ窓Ｎ −ＣＨ−Ｃ００Ｈ
（式中、Ｒは水酸基、メトキシ基、ジメチルアミノ基ま
たはジエチルアミノ基を示す、）しかしこの化合物もｒ
−ＧＴＰに対する基質反応性は、γ−Ｌ−グルタミルパ
ラニトロアニリドと大差がなく、基質反応性のよりすぐ
れた化合物が望まれていた。C) I! ■ H window N -CH-C00H (In the formula, R represents a hydroxyl group, a methoxy group, a dimethylamino group, or a diethylamino group.) However, this compound also has r
The substrate reactivity towards -GTP is not much different from that of γ-L-glutamylparanitroanilide, and a compound with better substrate reactivity has been desired.
一方、γ−Ｌ−グルタミルーｐ−（ジアルキルアミノ）
アニリンを基質とし、さらに酸化作用を有する物質と耐
拡散性カプラーを含有する多層分析要素は、特開昭５８
−１７５４９５号に開示されている。この分析要素は前
記特公昭５６−３５６６１号に記載された基質を用いて
いるため、Ｔ−ＧＴＰに対する基質反応の速度、従って
発色速度が不充分であった。On the other hand, γ-L-glutamyl-p-(dialkylamino)
A multilayer analytical element using aniline as a substrate and further containing an oxidizing substance and a diffusion-resistant coupler was disclosed in Japanese Patent Application Laid-open No. 58
-175495. Since this analytical element used the substrate described in Japanese Patent Publication No. 35661/1982, the rate of substrate reaction with T-GTP and, therefore, the rate of color development was insufficient.
さらに、分析要素の層中に耐拡散性カプラーを疎水性溶
媒を用いて分散するために製造工程が複雑であり、分散
のために有機溶剤や界面活性剤の使用を必要とした。Furthermore, the manufacturing process was complicated because the diffusion-resistant coupler was dispersed in the layer of the analytical element using a hydrophobic solvent, and required the use of organic solvents and surfactants for dispersion.
本発明の目的は、ｒ−ＧＴＰ活性値が短い時間で正確に
測定でき、かつ水添透性層中に疎水性溶媒を用いてカプ
ラーを分散することなく簡単な工程で製造することがで
きるｒ−ＧＴＰ活性測定用分析要素の提供にある。The purpose of the present invention is to enable the r-GTP activity value to be measured accurately in a short period of time, and to produce r-GTP in a simple process without using a hydrophobic solvent to disperse the coupler in the hydrogenated permeable layer. - To provide an analytical element for measuring GTP activity.
本発明の目的は、支持体上に少なくとも１ＩｌＩの試薬
層および少なくとも１層の展開層から成る分析素子にお
いて、該層の少なくとも１層中に酸化□作用のある物質
、カプラーおよび下記一般式〔！〕で示されるアニリド
誘導体またはその塩を含有する分析要素を用いることに
より達成された。The object of the present invention is to provide an analytical element comprising a reagent layer of at least 1IlI and at least one developing layer on a support, a substance having an oxidizing □ action, a coupler, and the following general formula [!] in at least one of the layers. ] was achieved by using an analytical element containing the anilide derivative or its salt.
式中、Ｒ，およびＲ３は、それぞれアルキル基またはヒ
ドロキシアルキル基を表わし、Ｒ１とＲ８が同時にアル
キル基をあられすことはないｅＲ３、Ｒａ 、Ｒｓおよ
びＲ６はそれぞれ水素原子、ハロゲン原子、ヒドロキシ
基、アミノ基、アルコキシ基、アシルアミド基、アリー
ルスルホンアミド基、アルキルスルホンアミド基または
アルキル基を表わす。In the formula, R and R3 each represent an alkyl group or a hydroxyalkyl group, and R1 and R8 cannot be an alkyl group at the same time.eR3, Ra, Rs, and R6 each represent a hydrogen atom, a halogen atom, a hydroxy group, It represents an amino group, an alkoxy group, an acylamido group, an arylsulfonamide group, an alkylsulfonamide group, or an alkyl group.
前記式中、Ｒ１またはＲ工で表わされるアルキルおよび
ヒドロキシアルキル基、炭素原子数１乃至４のものが好
ましい、アルキル基として例えばメチル基、エチル基、
ブチル基を挙げることができる。アルキル基は置換基を
有していてもよく、置換基としては例えばウレイド基、
テトラヒドロフリル基、カルボキシル基、メタンスルホ
ンアミド基、スルホ基、メトキシ基、エトキシ基、メト
キシエトキシ基、メトキシエトキシエトキシ基、メトキ
シテトラエトキシ基が挙げられるが、無置換アルキル基
が好ましい、ヒドロキシアルキル基の例は２−ヒドロキ
シエチル基、２−ヒドロキシ−１−メチルエチル基であ
る。Ｒ１およびＲｓとしては水素原子が好ましい、アル
キル基である場合、メチル基が好ましい、また、−１式
で示される化合物の塩としては、ｐ−１−ルエンスルホ
ン酸、スルホン酸、スルフィン酸、硫酸エステル、スル
ファミン酸、千オ硫酸Ｓ−エステル、カルボン酸、リン
酸エステル、アミドリン酸、リン酸、亜リン酸エステル
、有機ホウ素化合物、塩酸および硫酸等の有機酸または
無機酸の塩を挙げることができ以下に本発明に係るアニ
リド誘導体の代表的具体例を示すが、本発明は、これに
限定されるものではない。In the above formula, the alkyl and hydroxyalkyl groups represented by R1 or R are preferably those having 1 to 4 carbon atoms, and examples of the alkyl group include methyl group, ethyl group,
Mention may be made of the butyl group. The alkyl group may have a substituent, such as a ureido group,
Examples of the hydroxyalkyl group include a tetrahydrofuryl group, a carboxyl group, a methanesulfonamide group, a sulfo group, a methoxy group, an ethoxy group, a methoxyethoxy group, a methoxyethoxyethoxy group, and a methoxytetraethoxy group, and an unsubstituted alkyl group is preferred. Examples are 2-hydroxyethyl, 2-hydroxy-1-methylethyl. R1 and Rs are preferably hydrogen atoms, and when they are alkyl groups, methyl groups are preferred; salts of the compound represented by formula -1 include p-1-luenesulfonic acid, sulfonic acid, sulfinic acid, sulfuric acid; Mention may be made of salts of organic or inorganic acids such as esters, sulfamic acids, S-esters of 1,000 sulfates, carboxylic acids, phosphoric esters, amidophosphoric acids, phosphoric acids, phosphorous esters, organic boron compounds, hydrochloric acid and sulfuric acid. Representative examples of the anilide derivatives according to the present invention are shown below, but the present invention is not limited thereto.
１−（Ｌ−７−グルタミル）−４−（Ｎ−エチル−Ｎ−
（２−ヒドロキシエチル）アミノ）アニリド
（＋−２）
１−（Ｌ−ｒ−グルタミル）−４−（Ｎ−メチル−Ｎ−
（２−ヒドロキシエチル）アミノ）アニリド
１−（Ｌ−γ−グルタミル）−４−（Ｎ−エチル−Ｎ−
（２−ヒドロキシエチル）アミノ）−２−メチルアニリ
ド
１− （Ｌ−ｙ−グルタミル）−２−エチル−４−（Ｎ
−エチル−Ｎ−（２−ヒドロキシエチル）アミノ）アニ
リド
１−（Ｌ−ｒ−グルタミル）−４−（Ｎ、Ｎ−ジ（２−
ヒドロキシエチル）アミノ）アニリド１−（Ｌ−ｒ−グ
ルタミル）−４−（Ｎ、Ｎ−ジ（２−ヒドロキシエチル
）アミノ）−２−メチルアニリド
１−（Ｌ−γ−グルタミル）−４−（Ｎ−（２−ヒドロ
キシエチル）−Ｎ−（２−メトキシエチル）アミノ）ア
ニリド
（［−８）
１−（Ｌ−ｙ−グルタミル）−４−（Ｎ−エチル−Ｎ−
（２−ヒドロキシ−１−メチルエチル）アミノ）アニリ
ド
１−（Ｌ−γ−グルタミル）−４−（Ｎ、Ｎ−ジ（３−
ヒドロキシプロピル）アミノ）アニリド１−（Ｌ−ｙ−
グルタミル）−４−（Ｎ−エチル）−Ｎ−（２−ヒドロ
キシエチル）アミノ）−３−メチルアニリド
本発明に係るアニリド誘導体の塩は水溶性であり、水も
しくはバッファ水溶液中に容易に溶解して分析要素中に
添加できる。1-(L-7-glutamyl)-4-(N-ethyl-N-
(2-hydroxyethyl)amino)anilide (+-2) 1-(L-r-glutamyl)-4-(N-methyl-N-
(2-hydroxyethyl)amino)anilide 1-(L-γ-glutamyl)-4-(N-ethyl-N-
(2-hydroxyethyl)amino)-2-methylanilide 1-(L-y-glutamyl)-2-ethyl-4-(N
-ethyl-N-(2-hydroxyethyl)amino)anilide 1-(L-r-glutamyl)-4-(N,N-di(2-
hydroxyethyl)amino)anilide 1-(L-r-glutamyl)-4-(N,N-di(2-hydroxyethyl)amino)-2-methylanilide 1-(L-γ-glutamyl)-4-( N-(2-hydroxyethyl)-N-(2-methoxyethyl)amino)anilide ([-8) 1-(L-y-glutamyl)-4-(N-ethyl-N-
(2-hydroxy-1-methylethyl)amino)anilide 1-(L-γ-glutamyl)-4-(N,N-di(3-
Hydroxypropyl)amino)anilide 1-(L-y-
glutamyl)-4-(N-ethyl)-N-(2-hydroxyethyl)amino)-3-methylanilide The salt of the anilide derivative according to the present invention is water-soluble and easily dissolved in water or an aqueous buffer solution. can be added to the analytical element.
本発明の分析要素におけるアニリド誘導体およびその塩
の含有量は、その測定範囲領域（ダイナミックレンジ）
を決める上で重要であり、通常ｌポ中に０．５ｍＭ〜７
０５１Ｍ含有していれば良好な測定が可能であるが、好
ましくは１．０ｓＭ〜４０−一を用いる。The content of the anilide derivative and its salt in the analytical element of the present invention is determined by its measurement range (dynamic range).
It is important in determining the
If it contains 051M, good measurement is possible, but preferably 1.0sM to 40-1 is used.
本発明で用いるカプラーは、本発明のアニリド誘導体よ
り生成されるアニリン誘導体の酸化物とカップリング反
応して色素を生成する物質を言い、分子を耐拡散性にす
る大きさおよび立体配置を有する置換基（バラスト基と
呼ばれている）を分子中に有しない、フェノール類、ナ
フトール類、アセトアミド類およびピラゾロン化合物を
用いることができる。The coupler used in the present invention refers to a substance that generates a dye by a coupling reaction with the aniline derivative oxide produced from the anilide derivative of the present invention, and is a substituted substance having a size and configuration that makes the molecule resistant to diffusion. Phenols, naphthols, acetamides, and pyrazolone compounds that do not have groups (called ballast groups) in their molecules can be used.
以下に本発明に用いられるカプラーの代表的具体例を示
す。Typical specific examples of couplers used in the present invention are shown below.
１−ヒドロキシ−２−ナフトアミド
１− （２，４，６−ドリクロロフエニル）−３−ベン
ズアミド−５−ピラゾロン
１−（２，４，６−）ジクロロフェニル）−３−アニリ
ノ−５−ピラゾロン
２、 ４−ジクロロ−３−メチルフェノール２−クロロ
−３−メチル−４−メトキシカルボニルメトキシフェノ
ール
２−クロロ−３−メチル−４−カルボキシメトキシフェ
ノール
２．４−ジクロロ−３−メチル−６−ペンズアミドフェ
ノール
２．４−ジブロム−３−メチルフェノール２−エチルカ
ルボニルアミノフェノール２−エチルカルボニルアミノ
−５−メチルフェノール
２−フェニル−ジ−フルオルメチル−４−クロロフェノ
ール
２−メチル−４−メタンスルホンアミドフェノール
α−ベンゾイル−２−クロロアセトアニリド（２−１４
） ２−ヒドロキシベンズアミド（２−１５）
２−アセチルアミノフェノール（２−１６） ３
−アセチルアミノフェノール（２−１７） ２． ５
−ジクロロフェノール（２−１８） オルトクレゾー
ル
（２−１９） メタクレゾール
（２−２０） ２−クロロフェノール（２−２１）
３−クロロフェノール（２−２２） ２−メ
トキシフェノール（２−２３） ３−メトキシフェ
ノール（２−２４） ３−アセチルアミノ−５−メ
チルフッ −ル
（２−２５） ３−エチルカルボニルアミノフェノ
ール
（２−２６） フェノール
（２−２７） ｌ−ヒドロキシ−Ｎ−メチル−２−
ナフトアミド
上記のうち好ましいのは化合物（２−１６）および（２
−２５）である。1-hydroxy-2-naphthamide 1-(2,4,6-drichlorophenyl)-3-benzamido-5-pyrazolone 1-(2,4,6-dichlorophenyl)-3-anilino-5-pyrazolone 2, 4-dichloro-3-methylphenol 2-chloro-3-methyl-4-methoxycarbonylmethoxyphenol 2-chloro-3-methyl-4-carboxymethoxyphenol 2.4-dichloro-3-methyl-6-penzamidophenol 2.4-dibromo-3-methylphenol 2-ethylcarbonylaminophenol 2-ethylcarbonylamino-5-methylphenol 2-phenyl-di-fluoromethyl-4-chlorophenol 2-methyl-4-methanesulfonamidophenol α- Benzoyl-2-chloroacetanilide (2-14
) 2-hydroxybenzamide (2-15)
2-acetylaminophenol (2-16) 3
-acetylaminophenol (2-17) 2. 5
-Dichlorophenol (2-18) Orthocresol (2-19) Metacresol (2-20) 2-chlorophenol (2-21)
3-chlorophenol (2-22) 2-methoxyphenol (2-23) 3-methoxyphenol (2-24) 3-acetylamino-5-methylfluor (2-25) 3-ethylcarbonylaminophenol (2 -26) Phenol (2-27) l-hydroxy-N-methyl-2-
Among the above naphthamides, preferred are compounds (2-16) and (2
-25).
本発明の分析要素中にカプラーは１ｄ当り１から２０Ｉ
ＩＭ含むのが適当である。３〜１２＋ｍＭが好ましい、
カプラーは２種以上を併用してもよい。The couplers in the analytical elements of the invention are from 1 to 20 I/d.
It is appropriate to include IM. 3-12+mM is preferred.
Two or more couplers may be used in combination.
本発明の分析要素に用いる酸化作用のある物質は、公知
のものから選択することができる０例えばフェリシアン
化カリウム（赤血塩）、ヨウ素酸カリウム、過硫酸塩等
を用いることができる。フェリシアン化カリウム又はナ
トリウムが好ましい。The oxidizing substance used in the analytical element of the present invention can be selected from known substances, such as potassium ferricyanide (red blood salt), potassium iodate, persulfate, and the like. Potassium or sodium ferricyanide is preferred.
酸化作用のある物質は分析要素中にｉｎ？当り０．３か
ら３０ｍＭ含むのが適当で、１〜１０ｗＭが好ましい。Is there an oxidizing substance in the analysis element? It is appropriate to contain 0.3 to 30mM, preferably 1 to 10wM.
本発明に係る分析要素は少くとも一つの水滲透性層を有
し、分析すべき水性液体試料の成分（被検物質）と反応
して色素を生成する試薬として、前述のアニリド誘導体
またはその塩、カプラーおよび酸化作用のある物質を水
滲透性層に含有する。The analytical element according to the present invention has at least one water-permeable layer, and uses the above-mentioned anilide derivative or its salt as a reagent that reacts with a component (test substance) of an aqueous liquid sample to be analyzed to produce a dye. , a coupler and an oxidizing substance in the water-permeable layer.
本発明のアニリド誘導体から生成したアニリン誘導体を
酸化作用のある物質の作用により酸化する。The aniline derivative produced from the anilide derivative of the present invention is oxidized by the action of an oxidizing substance.
その結果、生じた酸化体がカプラーとカップリング反応
して、色素を生成する。As a result, the resulting oxidant undergoes a coupling reaction with the coupler to produce a dye.
本発明において酸化作用のある物質、カプラーおよび一
般式で示されるアニリド誘導体（またはその塩）は本発
明の分析素子の異なる層に別々に含有されてもよく、ま
た同一層に含有されても良い。In the present invention, the oxidizing substance, the coupler, and the anilide derivative (or its salt) represented by the general formula may be contained separately in different layers of the analytical element of the present invention, or may be contained in the same layer. .
本発明の分析要素は多孔性液体展開層、反応試薬層のほ
か、支持体、検出層、光遮蔽層、接着層、ろ過層、吸水
層、下塗り層および公知のその他の層を含む多重層の構
成を有してもよい、かような分析要素として、米国特許
第３．９９２，１５８号、同４，０４２，３３５号およ
び特開昭５５−１６４３５６号各明細書に開示されたも
のがある。The analytical element of the present invention has multiple layers including a porous liquid spreading layer, a reactive reagent layer, a support, a detection layer, a light shielding layer, an adhesive layer, a filtration layer, a water absorption layer, an undercoat layer, and other known layers. Examples of such analysis elements that may have the configuration include those disclosed in U.S. Pat. No. 3,992,158, U.S. Pat. .
光透過性支持体を用いる場合、本発明の乾式分析要素の
実用的に採りうる構成は、例えば（１１支持体上に試薬
層、その上に液体展開層を有するもの。When a light-transmitting support is used, a practical configuration of the dry analytical element of the present invention is, for example (11) having a reagent layer on the support and a liquid spreading layer thereon.
（２）支持体上に検出層、試薬層、液体展開層をこの順
に有するもの。(2) A device having a detection layer, a reagent layer, and a liquid development layer in this order on a support.
（３）支持体上に試薬層、光反射層、液体展開層をこの
１頓に有するもの。(3) One that has a reagent layer, a light reflection layer, and a liquid development layer all in one on a support.
（４）支持体上に検出層、試薬層、光反射層、液体展開
層をこの順に有するもの。(4) A support having a detection layer, a reagent layer, a light reflection layer, and a liquid development layer in this order.
（５） 支持体上に検出層、光反射層、試薬層、液体
展開層をこの順に有するもの。(5) A support having a detection layer, a light reflection layer, a reagent layer, and a liquid development layer in this order.
支持体と試薬層との間に吸水層を設けてもよい。A water absorption layer may be provided between the support and the reagent layer.
上記（１）ないしく３）において試薬層と検出層または
液体展開層の間にろ過層を設けてもよい、上記（３）な
いしく５）において光反射層と検出層、試薬層または液
体展開層との間、試薬層と検出層または液体展開層との
間に、さらにろ過層を設けてもよい。In (1) to 3) above, a filtration layer may be provided between the reagent layer and the detection layer or the liquid development layer, and in (3) to 5) above, the light reflection layer and the detection layer, the reagent layer or the liquid development layer may be provided. A filtration layer may be further provided between the reagent layer and the detection layer or the liquid spreading layer.
本発明の乾式分析要素の試薬層としては、親水性ポリマ
ーを結合剤とする実質的に均一の層または特開昭５８−
７０１６３号に記載されたような多孔性層が好適である
。親水性ポリマーとして例えば、ゼラチンおよびこれら
の誘導体（例えばフタル化ゼラチン）、セルロース誘導
体（例えばヒドロキシメチルセルロース）、アガロース
、アクリルアミド重合体、メタアクリルアミド重合体、
アクリルアミドまたはメタアクリルアミドと各種ビニル
性モノマーとの共重合体等が利用できる。The reagent layer of the dry analytical element of the present invention may be a substantially uniform layer containing a hydrophilic polymer as a binder or
Porous layers such as those described in US Pat. No. 70,163 are preferred. Examples of hydrophilic polymers include gelatin and derivatives thereof (e.g. phthalated gelatin), cellulose derivatives (e.g. hydroxymethyl cellulose), agarose, acrylamide polymers, methacrylamide polymers,
Copolymers of acrylamide or methacrylamide and various vinyl monomers can be used.
試薬層には酵素に対する基質、酸化剤、カプラー、緩衝
剤等を含有させることができる。The reagent layer can contain a substrate for the enzyme, an oxidizing agent, a coupler, a buffer, and the like.
本発明の分析要素の試薬層に含有させることができる緩
衝剤の例としては、炭酸塩、ホウ酸塩、燐酸塩やグツド
（Ｇｏｏｄ）の緩衝剤などを挙げることができる。これ
らの緩衝剤は「蛋白質・酵素の基礎実験法ｊ （堀尾武
−ほか著、南江堂、１９８１年） 、Ｂｉｏｃｈｅｓ＋
１ｓｔｒｙ 誌 第５Ｓ 第２号第４６７ページより
第４７７ページ（１９６６年）等の文献を参考にして選
択することができる。Examples of buffers that can be contained in the reagent layer of the analytical element of the present invention include carbonate, borate, phosphate and Good's buffers. These buffers are described in "Basic Experimental Methods for Proteins and Enzymes (Takeshi Horio et al., Nankodo, 1981)," Bioches+
The selection can be made by referring to literature such as 1stry Magazine No. 5S No. 2, pages 467 to 477 (1966).
多孔性液体展開層は、液体計量作用を有する展開層であ
ることが好ましい。液体計量作用を有するとは、その表
面に点着供給された液体試料を、その中に含有している
部分を実質的に偏在させることなく、横（水平）方向に
単位面積当りほぼ一定量の割合で広げる作用を有するこ
とである。Preferably, the porous liquid spreading layer is a spreading layer having a liquid metering function. Having a liquid metering effect means that the liquid sample that is dotted onto the surface of the sample is distributed in an approximately constant amount per unit area in the lateral (horizontal) direction without substantially unevenly distributing the liquid sample contained therein. It has the effect of expanding the ratio.
展開層を構成する材料としては、濾紙、不織布、織物生
地（例えば平織生地）、編物生地（例えば、トリコット
編）、ガラス繊維濾紙等を用いることができる。これら
のうちでは、織物生地および編物生地等が好ましい。織
物等は特開昭５７−６６３５９号に記載されたようなグ
ロー放電処理をしてもよい、展開層には、展開面積、展
開速度等を調節するため、特開昭６０−２２２７７０号
、特願昭６１−１２２８７５号、６１−１２２８７６号
、６１−１４３７５４号に記載したような親水性高分子
あるいは界面活性剤を含有してもよい。As the material constituting the spreading layer, filter paper, nonwoven fabric, woven fabric (for example, plain weave fabric), knitted fabric (for example, tricot fabric), glass fiber filter paper, etc. can be used. Among these, woven fabrics, knitted fabrics, etc. are preferred. Fabrics and the like may be subjected to glow discharge treatment as described in JP-A No. 57-66359. The spreading layer may be treated with glow discharge treatment as described in JP-A-60-222770, JP-A-60-222770, in order to adjust the spreading area, spreading speed, etc. It may contain a hydrophilic polymer or a surfactant as described in Japanese Patent Applications No. 61-122875, No. 61-122876, and No. 61-143754.
展開層を接着し積層するための接着層を、試薬層、光速
へい層、濾過層、吸水層、検出層等の層の上に設けても
よい、接着層は水で膨潤したときに展開層を接着するこ
とができるような親木性ポリマー、例えばゼラチン、ゼ
ラチン誘導体、ポリアクリルアミド等からなることが好
ましい。An adhesive layer for adhering and laminating the developing layer may be provided on layers such as a reagent layer, a light-transmitting layer, a filtration layer, a water absorption layer, a detection layer, etc. When the adhesive layer is swollen with water, the developing layer It is preferably made of a wood-philic polymer that can be used to adhere materials, such as gelatin, gelatin derivatives, polyacrylamide, and the like.
本発明の分析要素において、γ−ＧＴＰに対する基質は
試薬層に含んでもよいが、展開層に含まれるのが好まし
い、酸化剤、カプラーも展開層に含有させることができ
る。In the analytical element of the present invention, the substrate for γ-GTP may be contained in the reagent layer, but the oxidizing agent and coupler, which are preferably contained in the developing layer, can also be contained in the developing layer.
光ｉ繭層は、検出層、試薬層等に生じた検出可能な変化
（色変化、発色等）を光透過性を有する支持体側から反
射測光する際に、展開層に点着供給された被検液の色、
特に試料が全血である場合のヘモグロビンの赤色等を遮
蔽するとともに光反射層または背景層として機能する。The optical i-cocoon layer is a coating that is dotted onto the developing layer when measuring detectable changes (color change, color development, etc.) occurring in the detection layer, reagent layer, etc. from the light-transmitting support side. The color of the test solution,
Particularly when the sample is whole blood, it blocks the red color of hemoglobin and functions as a light reflecting layer or background layer.
光遮蔽層としては、皮膜形成能を有する親水性ポリマー
をバインダーとして、酸化チタン、硫酸バリウム等の光
反射性微粒子が分散された水浸透性の層が好ましい、バ
インダーとしてはゼラチン、ゼラチン誘導体、ポリアク
リルアミド等が好ましい０分析要素には、必要に応じ展
開層、試薬層、検出層等に酸化チタン等の光反射粒子を
含有させてもよい。The light shielding layer is preferably a water-permeable layer in which light-reflecting fine particles such as titanium oxide or barium sulfate are dispersed using a hydrophilic polymer having film-forming ability as a binder.As the binder, gelatin, gelatin derivatives, polyester, etc. The 0 analysis element, which is preferably acrylamide, may contain light reflecting particles such as titanium oxide in the developing layer, reagent layer, detection layer, etc., if necessary.
本発明の分析要素においては、特開昭６１−２９３３９
８号の記載のようにγ〜グルタミルバラニトロアニリド
を基質として用いるために界面活性剤で可溶化する操作
を必要としない。しかも、本発明で用いる基質は、γ−
グルタミルバラニトロアニリドや、特公昭５６−３５６
６１号発明の基質に比し基質反応性がすぐれるため、本
発明の分析要素によりγ−ＧＴＰ活性測定において高い
感度が得られる。In the analysis element of the present invention, Japanese Patent Application Laid-Open No. 61-29339
As described in No. 8, in order to use γ-glutamyl varanitroanilide as a substrate, it is not necessary to solubilize it with a surfactant. Moreover, the substrate used in the present invention is γ-
Glutamylvaranitroanilide, Special Publication 1986-356
Since the substrate reactivity is superior to that of the substrate of the No. 61 invention, the analytical element of the present invention provides high sensitivity in measuring γ-GTP activity.
本発明の分析要素においては、特開昭５８−１７５４９
５号のような耐拡散性カプラーを用いることなく、充分
に高い分析結果の信転性および正確性が得られる０分析
要素中にカプラーと酸化作用のある物質が含まれている
と、分析要素の保存中に望ましくない酸化による着色が
起こり、正確性を著しく悪化させることがあるが、本発
明のカプラーを用いる場合、分析要素の保存性はすぐれ
ており、長期間分析素子の信転性が維持される。In the analysis element of the present invention, Japanese Patent Application Laid-Open No. 58-17549
Sufficiently high reliability and accuracy of analysis results can be obtained without using a diffusion-resistant coupler such as No. 5.0 If the analytical element contains a coupler and a substance that has an oxidizing effect, Undesirable coloring due to oxidation may occur during storage, which can significantly deteriorate accuracy. However, when the coupler of the present invention is used, the analytical element has excellent storage stability and reliability of the analytical element over a long period of time. maintained.
検体中の妨害物質、特にアルコルビン酸等の還元物質に
対する影響も小さい。The effect on interfering substances in the specimen, especially reducing substances such as ascorbic acid, is also small.
〔実施例１〕
透明ポリエステルフィルムを支持体とするγ−グルタミ
ルトランスフェラーゼ活性測定用乾式多層分析要素を、
下記のようにして作製した。[Example 1] A dry multilayer analytical element for measuring γ-glutamyltransferase activity using a transparent polyester film as a support,
It was produced as follows.
Ａ層（第１試薬層）：
下記組成の溶液を、厚さ１５０μｍの透明ポリエステル
フィルム上に、乾燥時の厚さ１５μｍになるように塗布
乾燥した。Layer A (first reagent layer): A solution having the following composition was applied onto a 150 μm thick transparent polyester film and dried to a dry thickness of 15 μm.
水 １３０Ｊ
！ゼラチン ２０ｇフェリシ
アン化カリウム １．５ｇトリス（ヒドロ
キシメチル）
アミノメタン ０．６ｇ（溶解後左
性ソーダ水溶液でｐＨ８，３に調整）Ｂ層（第２試薬層
）：
下記組成の溶液を調製し、上記Ａ層上に乾燥時の厚さ１
５μｍになるよう塗布・乾燥した。Water 130J
! Gelatin 20g Potassium ferricyanide 1.5g Tris(hydroxymethyl) aminomethane 0.6g (After dissolving, adjust the pH to 8.3 with aqueous sodium chloride solution) B layer (second reagent layer): Prepare a solution with the following composition, and use the above A Dry thickness 1 on layer
It was coated and dried to a thickness of 5 μm.
水 １３０．
／ゼラチン ２０ｇｍ−アセ
チルアミノフェノール １．５ｇトリス（ヒドロキ
シメチル）
アミノメタン １．２ｇ（溶解後力
性メーダ水溶液でｐＨ８，３に調整）０層（展開層）
：
下記組成の溶液を調製し酢酸セルロースから成るメンプ
ラン・フィルター（富士写真フィルム■製 フジミクロ
フィルターＦＭ −３００） ニ含ｔ＋させ、乾燥させ
た。Water 130.
/ Gelatin 20gm - Acetylaminophenol 1.5g Tris(hydroxymethyl) Aminomethane 1.2g (After dissolving, adjust the pH to 8.3 with an aqueous solution of Meida) 0 layer (Development layer)
A solution having the following composition was prepared, mixed with a Menpuran filter (Fuji Micro Filter FM-300 manufactured by Fuji Photo Film) made of cellulose acetate, and dried.
水 ３００Ｊ
ポリアクリルアミド
（平均分子量４万） １０ｇγ−Ｌ−
グルタミルーｐ−（Ｎ。Water 300J
Polyacrylamide (average molecular weight 40,000) 10gγ-L-
Glutamyl p-(N.
Ｎ−ジ（２−ヒドロキシエチ
ル）アミノコアニリド ５ｇグリシルグリ
シン １０ｇトリス（ヒドロキシメチ
ル）
アミノメタン ４ｇ（溶解後、力
性ソーダでｐＨ８，３に調製した）。N-di(2-hydroxyethyl)aminocoanilide 5g glycylglycine 10g tris(hydroxymethyl)aminomethane 4g (after dissolving, the pH was adjusted to 8.3 with sodium hydroxide).
少量の水で湿した前記Ｂ層の上に、上記展開層を積層・
固定（ラミネート）シた。最後に全体を充分に乾燥させ
ることにより、γ−グルタミルトランスフェラーゼ活性
測定用乾式分析要素（フィルム）を完成した。The above development layer is laminated on the B layer moistened with a small amount of water.
Fixed (laminate). Finally, by thoroughly drying the whole, a dry analytical element (film) for measuring γ-glutamyl transferase activity was completed.
この分析要素にＴ−グルタミルトランスフェラーゼ１５
００単位／ｌを含む液１０μｌを点着し３７℃に２ない
し５分間加温することにより、６２５ｎｍに反射吸収極
大を有する青色の発色が得られた。This analytical element includes T-glutamyltransferase 15.
By spotting 10 μl of a solution containing 0.00 units/l and heating it to 37° C. for 2 to 5 minutes, a blue color having a reflection absorption maximum at 625 nm was obtained.
透明支持体側から反射測光したところ、反射光学濃度は 点着直後 ０．４９ ２分後 ０．９１ ５分後 １．２８ であった。Reflection photometry from the transparent support side revealed that the reflection optical density was Immediately after spotting 0.49 2 minutes later 0.91 5 minutes later 1.28 Met.
同様にＴ−グルタミルトランスフェラーゼ３０ないし２
０００単位／１を含む液１０μｌを点着し３７℃に２分
および５分間加温した後、それぞれ波長６２５ｎ−にお
いて反射測光したところ、２分後と５分後の光学濃度の
差は酵素活性にほぼ比例していた。Similarly, T-glutamyl transferase 30 to 2
After spotting 10 μl of a solution containing 1,000 units/1 and heating it at 37°C for 2 and 5 minutes, reflection photometry was performed at a wavelength of 625 nm. The difference in optical density after 2 and 5 minutes was determined by the enzyme activity. was almost proportional to.
（実施例２〕
実施例１におけるｒ−Ｌ−グルタミル−ｐ−（Ｎ、Ｎ−
ジ（２−ヒドロキシエチル）アミノコアニリドの代わり
にｙ−Ｌ−グルタミル−ｐ−〔Ｎ−エチル−Ｎ−（２−
ヒドロキシエチル）アミノコアニリドを用いたところ、
同様の結果が得られた。(Example 2) r-L-glutamyl-p-(N,N-
y-L-glutamyl-p-[N-ethyl-N-(2-
When using hydroxyethyl) aminocoanilide,
Similar results were obtained.
Claims (4)
とも１層の展開層を有する分析要素であって該層の少な
くとも１層に酸化作用のある物質、バラスト基を有しな
いカプラーおよび下記一般式〔 I 〕で示されるアニリ
ド誘導体またはその塩を含有することを特徴とするガン
マグルタミルトランスペプチダーゼ活性測定用分析要素
。 〔 I 〕▲数式、化学式、表等があります▼ （式中、Ｒ＿１およびＲ＿２は、それぞれアルキル基、
またはヒドロキシアルキル基を表わすが、Ｒ＿１Ｒ＿２
が同時にアルキル基を表わすことはない、Ｒ＿３、Ｒ＿
４、Ｒ＿５およびＲ＿６はそれぞれ水素原子、ハロゲン
原子、ヒドロキシ基、アミノ基、アルキル基、アルコキ
シ基、アシルアミド基、アリールスルホンアミド基、ま
たはアルキルスルホンアミド基を表わす。）(1) An analytical element having at least one reagent layer and at least one development layer on a support, at least one of which has an oxidizing substance, a coupler having no ballast group, and the following general formula: An analytical element for measuring gamma glutamyl transpeptidase activity, comprising an anilide derivative represented by [I] or a salt thereof. [I]▲Mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R_1 and R_2 are an alkyl group,
or represents a hydroxyalkyl group, R_1R_2
do not represent an alkyl group at the same time, R_3, R_
4, R_5 and R_6 each represent a hydrogen atom, a halogen atom, a hydroxy group, an amino group, an alkyl group, an alkoxy group, an acylamido group, an arylsulfonamide group, or an alkylsulfonamide group. )
＿２がヒドロキシアルキル基、Ｒ＿３がメチル基、Ｒ＿
４、Ｒ＿５およびＲ＿６がそれぞれ水素原子をあらわす
特許請求の範囲（１）の分析要素。(2) In general formula [I], R_1 is an alkyl group, R
_2 is a hydroxyalkyl group, R_3 is a methyl group, R_
4. The analytical element according to claim (1), wherein R_5 and R_6 each represent a hydrogen atom.
々ヒドロキシアルキル基、Ｒ＿３、Ｒ＿４、Ｒ＿５およ
びＲ＿６がそれぞれ水素原子をあらわす特許請求の範囲
（１）の分析要素。(3) The analytical element according to claim (1), wherein in the general formula [I], R_1 and R_2 each represent a hydroxyalkyl group, and R_3, R_4, R_5 and R_6 each represent a hydrogen atom.
ドロキシエチル基、Ｒ＿３、Ｒ＿４、Ｒ＿５およびＲ＿
６が水素原子をあらわす特許請求の範囲（１）の分析要
素。(4) In general formula [I], R_1 and R_2 are hydroxyethyl groups, R_3, R_4, R_5 and R_
The analytical element according to claim (1), wherein 6 represents a hydrogen atom.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5166187A JPS63216500A (en) | 1987-03-06 | 1987-03-06 | Analytic element for determination of enzymatic activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5166187A JPS63216500A (en) | 1987-03-06 | 1987-03-06 | Analytic element for determination of enzymatic activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63216500A true JPS63216500A (en) | 1988-09-08 |
Family
ID=12893058
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5166187A Pending JPS63216500A (en) | 1987-03-06 | 1987-03-06 | Analytic element for determination of enzymatic activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63216500A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006080329A1 (en) * | 2005-01-26 | 2006-08-03 | Fujifilm Corporation | Multilayer analysis element |
-
1987
- 1987-03-06 JP JP5166187A patent/JPS63216500A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006080329A1 (en) * | 2005-01-26 | 2006-08-03 | Fujifilm Corporation | Multilayer analysis element |
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