CN1760368A - Method for testing activity of 5'-nucleotidase, diagnosis kit for 5'-nucleotidase - Google Patents
Method for testing activity of 5'-nucleotidase, diagnosis kit for 5'-nucleotidase Download PDFInfo
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- CN1760368A CN1760368A CN 200410064911 CN200410064911A CN1760368A CN 1760368 A CN1760368 A CN 1760368A CN 200410064911 CN200410064911 CN 200410064911 CN 200410064911 A CN200410064911 A CN 200410064911A CN 1760368 A CN1760368 A CN 1760368A
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- acid
- phosphonuclease
- nucleotidase
- ethyl
- hydroxyl
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 102000004008 5'-Nucleotidase Human genes 0.000 title claims abstract description 22
- 108010043671 prostatic acid phosphatase Proteins 0.000 title claims abstract description 22
- 238000012360 testing method Methods 0.000 title description 15
- 238000003745 diagnosis Methods 0.000 title description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 48
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 38
- 229940107700 pyruvic acid Drugs 0.000 claims abstract description 19
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 15
- 108010042687 Pyruvate Oxidase Proteins 0.000 claims abstract description 15
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 15
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 46
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 30
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 26
- 238000013016 damping Methods 0.000 claims description 25
- 239000012530 fluid Substances 0.000 claims description 25
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims description 16
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims description 16
- 229940045145 uridine Drugs 0.000 claims description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- 238000009007 Diagnostic Kit Methods 0.000 claims description 12
- 229910019142 PO4 Inorganic materials 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 239000010452 phosphate Substances 0.000 claims description 12
- PQDSQOCJSMICOM-UHFFFAOYSA-N [Cl].OC1=CC=CC=C1 Chemical compound [Cl].OC1=CC=CC=C1 PQDSQOCJSMICOM-UHFFFAOYSA-N 0.000 claims description 10
- YNQLUTRBYVCPMQ-UHFFFAOYSA-N alpha-methyl toluene Natural products CCC1=CC=CC=C1 YNQLUTRBYVCPMQ-UHFFFAOYSA-N 0.000 claims description 10
- IHWDSEPNZDYMNF-UHFFFAOYSA-N 1H-indol-2-amine Chemical compound C1=CC=C2NC(N)=CC2=C1 IHWDSEPNZDYMNF-UHFFFAOYSA-N 0.000 claims description 9
- 230000001186 cumulative effect Effects 0.000 claims description 9
- 150000004060 quinone imines Chemical class 0.000 claims description 9
- 230000035484 reaction time Effects 0.000 claims description 9
- 229910052785 arsenic Inorganic materials 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 7
- NVPNUVHLQKUCPX-UHFFFAOYSA-N 2-bromo-3-hydroxybenzenesulfonic acid Chemical compound OC1=CC=CC(S(O)(=O)=O)=C1Br NVPNUVHLQKUCPX-UHFFFAOYSA-N 0.000 claims description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 6
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 6
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 claims description 6
- 229960004424 carbon dioxide Drugs 0.000 claims description 6
- 229910002090 carbon oxide Inorganic materials 0.000 claims description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 6
- XIMKEDRSBNHZHC-UHFFFAOYSA-N n-ethyl-n-(3-methylphenyl)hydroxylamine Chemical class CCN(O)C1=CC=CC(C)=C1 XIMKEDRSBNHZHC-UHFFFAOYSA-N 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 claims description 6
- WKOLLVMJNQIZCI-UHFFFAOYSA-N vanillic acid Chemical compound COC1=CC(C(O)=O)=CC=C1O WKOLLVMJNQIZCI-UHFFFAOYSA-N 0.000 claims description 6
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 claims description 6
- 239000001533 (4R,6S)-2,4,6-trimethyl-1,3,5-dithiazinane Substances 0.000 claims description 5
- CSQFODQOQLFYIN-UHFFFAOYSA-N 3-chloro-2-hydroxybenzenesulfonic acid Chemical class OC1=C(Cl)C=CC=C1S(O)(=O)=O CSQFODQOQLFYIN-UHFFFAOYSA-N 0.000 claims description 5
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 5
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 5
- RZXMPPFPUUCRFN-UHFFFAOYSA-N p-toluidine Chemical class CC1=CC=C(N)C=C1 RZXMPPFPUUCRFN-UHFFFAOYSA-N 0.000 claims description 5
- JJYPMNFTHPTTDI-UHFFFAOYSA-N 3-methylaniline Chemical compound CC1=CC=CC(N)=C1 JJYPMNFTHPTTDI-UHFFFAOYSA-N 0.000 claims description 4
- WGYQCZSZSXMRIZ-UHFFFAOYSA-N C(C=1C(O)=CC=CC1)(=O)O.[Br] Chemical compound C(C=1C(O)=CC=CC1)(=O)O.[Br] WGYQCZSZSXMRIZ-UHFFFAOYSA-N 0.000 claims description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 229930010555 Inosine Natural products 0.000 claims description 3
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims description 3
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 3
- 229960003786 inosine Drugs 0.000 claims description 3
- 229960004418 trolamine Drugs 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 150000005846 sugar alcohols Polymers 0.000 claims description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims 2
- 229940058020 2-amino-2-methyl-1-propanol Drugs 0.000 claims 1
- PDLNHDSYGLTYDS-UHFFFAOYSA-N 3-aminopropanoic acid;hydrochloride Chemical compound Cl.NCCC(O)=O PDLNHDSYGLTYDS-UHFFFAOYSA-N 0.000 claims 1
- 108010008488 Glycylglycine Proteins 0.000 claims 1
- CBTVGIZVANVGBH-UHFFFAOYSA-N aminomethyl propanol Chemical compound CC(C)(N)CO CBTVGIZVANVGBH-UHFFFAOYSA-N 0.000 claims 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims 1
- 235000011130 ammonium sulphate Nutrition 0.000 claims 1
- 229940043257 glycylglycine Drugs 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- 238000005859 coupling reaction Methods 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 abstract description 2
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 239000003381 stabilizer Substances 0.000 abstract 1
- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 6
- 150000003016 phosphoric acids Chemical class 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- -1 3-sulfopropyl Chemical group 0.000 description 2
- 206010008635 Cholestasis Diseases 0.000 description 2
- AFBPFSWMIHJQDM-UHFFFAOYSA-N N-methylaniline Chemical compound CNC1=CC=CC=C1 AFBPFSWMIHJQDM-UHFFFAOYSA-N 0.000 description 2
- 231100000359 cholestasis Toxicity 0.000 description 2
- 230000007870 cholestasis Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
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- 238000005259 measurement Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- PHOLIFLKGONSGY-CSKARUKUSA-N (e)-(3-methyl-1,3-benzothiazol-2-ylidene)hydrazine Chemical compound C1=CC=C2S\C(=N\N)N(C)C2=C1 PHOLIFLKGONSGY-CSKARUKUSA-N 0.000 description 1
- VXEQGQXRKQSAMW-UHFFFAOYSA-N 2-amino-2-methylpropan-1-ol Chemical compound CC(C)(N)CO.CC(C)(N)CO VXEQGQXRKQSAMW-UHFFFAOYSA-N 0.000 description 1
- DEENCNYPFDWDSA-UHFFFAOYSA-N 2-ethyl-1,3-benzothiazole-6-sulfonic acid Chemical compound C1=C(S(O)(=O)=O)C=C2SC(CC)=NC2=C1 DEENCNYPFDWDSA-UHFFFAOYSA-N 0.000 description 1
- ZEYHEAKUIGZSGI-UHFFFAOYSA-N 4-methoxybenzoic acid Chemical compound COC1=CC=C(C(O)=O)C=C1 ZEYHEAKUIGZSGI-UHFFFAOYSA-N 0.000 description 1
- QZHXKQKKEBXYRG-UHFFFAOYSA-N 4-n-(4-aminophenyl)benzene-1,4-diamine Chemical compound C1=CC(N)=CC=C1NC1=CC=C(N)C=C1 QZHXKQKKEBXYRG-UHFFFAOYSA-N 0.000 description 1
- MBGYSHXGENGTBP-UHFFFAOYSA-N 6-(2-ethylhexoxy)-6-oxohexanoic acid Chemical compound CCCCC(CC)COC(=O)CCCCC(O)=O MBGYSHXGENGTBP-UHFFFAOYSA-N 0.000 description 1
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N Aminoantipyrine Natural products CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 1
- AHPQKAVAXVYVAZ-UHFFFAOYSA-N BrC=1C(=C(C(=C(C(=O)O)C1)O)Br)Br.OC1=C(C(=O)O)C=CC=C1.[Br] Chemical compound BrC=1C(=C(C(=C(C(=O)O)C1)O)Br)Br.OC1=C(C(=O)O)C=CC=C1.[Br] AHPQKAVAXVYVAZ-UHFFFAOYSA-N 0.000 description 1
- HPJUARPONYNFMA-UHFFFAOYSA-N C(C)N(C1=CC(=CC=C1)C)CC.C1(=CC(=CC=C1)N)C Chemical compound C(C)N(C1=CC(=CC=C1)C)CC.C1(=CC(=CC=C1)N)C HPJUARPONYNFMA-UHFFFAOYSA-N 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- PGXABRVTTLRYCN-UHFFFAOYSA-N Cl.Cl.Cl.Cl.C(=O)(O)CCN Chemical compound Cl.Cl.Cl.Cl.C(=O)(O)CCN PGXABRVTTLRYCN-UHFFFAOYSA-N 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 208000024815 Granulomatous liver disease Diseases 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 206010023129 Jaundice cholestatic Diseases 0.000 description 1
- JLTDJTHDQAWBAV-UHFFFAOYSA-N N,N-dimethylaniline Chemical compound CN(C)C1=CC=CC=C1 JLTDJTHDQAWBAV-UHFFFAOYSA-N 0.000 description 1
- ZQYUHRVUJCLGRX-UHFFFAOYSA-N NCC(=O)NCC(O)=O.NCC(=O)NCC(O)=O Chemical compound NCC(=O)NCC(O)=O.NCC(=O)NCC(O)=O ZQYUHRVUJCLGRX-UHFFFAOYSA-N 0.000 description 1
- 201000005267 Obstructive Jaundice Diseases 0.000 description 1
- GRPYQYXQLBQRJZ-UHFFFAOYSA-N [Na].Cc1cccc(NCC(O)CS(O)(=O)=O)c1 Chemical compound [Na].Cc1cccc(NCC(O)CS(O)(=O)=O)c1 GRPYQYXQLBQRJZ-UHFFFAOYSA-N 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229950006790 adenosine phosphate Drugs 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- 208000003167 cholangitis Diseases 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 231100000843 hepatic granuloma Toxicity 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- ILUJQPXNXACGAN-UHFFFAOYSA-N ortho-methoxybenzoic acid Natural products COC1=CC=CC=C1C(O)=O ILUJQPXNXACGAN-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- NMWCVZCSJHJYFW-UHFFFAOYSA-M sodium;3,5-dichloro-2-hydroxybenzenesulfonate Chemical compound [Na+].OC1=C(Cl)C=C(Cl)C=C1S([O-])(=O)=O NMWCVZCSJHJYFW-UHFFFAOYSA-M 0.000 description 1
- MWFOPMKUGZLPQA-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)propane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CCCN(CC)C1=CC=CC(OC)=C1 MWFOPMKUGZLPQA-UHFFFAOYSA-M 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
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- 230000017105 transposition Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A reagent kit for diagnosing 5'-nucleotidase is composed of buffer liquid, uridine monophosphate, pyruvic acid, pyruvate oxidase, peroxidase, stabilizer and reduced chromogen group. A process for measuring the activity of 5'-nucleotidase includes such steps as proportionally mixing specimen with reagents, enzyme coupling reaction and biochemically analyzing the final resultant to determine the variation in light absorptivity of master wavelength and in turn the activity of 5'-nucleotidase. Its advantages are high sensitivity and precision, and no pollution.
Description
Technical field
The present invention relates to a kind ofly to measure 5 '-method of activity of 5 '-nucleotidase, the invention still further relates to simultaneously be used to realize 5 of this method '-the phosphonuclease diagnostic kit, belong to medical test determination techniques field.
Background technology
Medical research shows, 5 '-phosphonuclease (5NT) increases and is mainly seen in the obstructive jaundice, also is found in liver cancer and hepatitis.When the concurrent cholangitis of cholestasis, primary and Secondary cases cholehepatocirrhosis and chronic hepatitis, the 5NT rate of rise is higher than alkaline phosphatase; The susceptibility that 5NT raises when liver tumor and hepatic granuloma is higher than alkaline phosphatase.Because 5 '-activity of 5 '-nucleotidase do not have physiological and raise, and is not only responsive than alkaline phosphatase for diagnosis infant's hepatopathy and gestational liver function cholestasis, and specificity is arranged.Therefore measure 5 '-activity of phosphonuclease is significant for the diagnosis of disease.
5 '-activity determination method of phosphonuclease is a lot, mainly contains isotope substrate method, chemical phosphorus acid test method (nineteen twenty-five).Understand according to the applicant, generally adopt the isotropic substance Substrate test method at present in the world, method is: 5 '-phosphonuclease acts on H
3-dUMP, after the termination reaction,, try to achieve 5 by the ion exchange column analytical results '-activity of 5 '-nucleotidase.Perhaps utilize 5 '-phosphonuclease produces inorganic phosphorus after acting on single adenosine phosphate, analyze content of inorganic phosphorus with Chemical acid method again and calculated 5 '-activity of phosphonuclease.
Method of isotope substrate is complicated to also have isotopic contamination, and needs Isotope analyzer, therefore is difficult to apply conscientiously.Determination of inorganic phosphorus is the method for nineteen twenty-five invention, and accuracy is bad, and strong acid contaminate environment or the like also is not suitable for applying.
Summary of the invention
Propose a kind of can overcome 5 of above prior art shortcoming '-measuring method of activity of 5 '-nucleotidase, provide simultaneously in order to realize 5 of this method '-the phosphonuclease diagnostic kit.Adopt reagent in this test kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out 5 '-activity of 5 '-nucleotidase is measured, and finding speed is fast, accuracy is high, thereby can obtain practical applying.
The present invention measures 5 '-step of activity of 5 '-nucleotidase is as follows:
1), with sample and the reagent mix of mainly forming by uridine list phosphoric acid, pyruvic acid, pyruvic oxidase, peroxidase, make it to take place the reaction of following method principle:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+two
Carbonoxide+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination
PeroxidaseThe indoleamine chromogen or
Quinone-imine chromogen+water
2), detect the end reaction thing in the speed that predominant wavelength 400--600nm absorbancy rises, calculate 5 '-the active size of phosphonuclease.
Usually step 2) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detect the speed that predominant wavelength 400--600nm absorbancy rises, calculate 5 '-the active size of phosphonuclease.
The blending ratio of above sample and reagent by volume is 1/10 to 1/250, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction times was controlled at conventional 2-30 minute.
The present invention uses 5 '-enzyme linked reaction such as phosphonuclease coupling pyruvic oxidase, peroxidase, with uridine list phosphoric acid is substrate, and reduced form chromogen (Chromogen) combined system, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen (Quioneimine) or indoleamine chromogen (Indamine) dyestuff, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect 5 '-size of activity of 5 '-nucleotidase.The advantage of this enzyme linked reaction system has: (1) its utilize will be colourless the reduced form chromogen make up be oxidized to coloured quinone-imine chromogen or indoleamine chromogen dyestuff and reflect 5 '-activity of 5 '-nucleotidase, the good advantage of tolerance range is arranged.(2) reacted constituent of this enzyme linked reaction system composition all adds, and is not subjected to the pollution of inside and outside source material, and test result is accurate.
In order to realize 5 of the inventive method '-the phosphonuclease diagnostic kit can be single agent, comprising:
Damping fluid 40--200mmol/l
Uridine list phosphatase 11--50mmol/l
Pyruvic acid 1--20mmol/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--50000U/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination 0.1--20mmol/l
3-methyl-2-Proxel hydrazone (3-methyl-2-benzothiazolinone-hydrszone is called for short MBTH) that above reduced form chromogen combination can be 0.1-20mmol/l or the amino anti-arsenic of 4-(4-aminoantipyrine) of 0.1-20mmol/l combine with one of following 14 kinds of compositions of 0.1--20mmol/l:
PHENOL 99.8 MIN ((CARBOLIC ACID)) (phenol)
N-ethyl-N-(3-thiopropyl)-m-thialdine amine (N-ethyl-N-(3-sulfopropyl)
-m-anisidine is called for short ESPAS)
N, and the two ethyls of N--m-Tolylamine (N, N-Diethyl-m-toluidine)
2, and the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-(2,4-Dichloriphenol)
2,4, and 6-three bromo-3-hydroxyl-Phenylsulfonic acid (2,4,6-Tribromo-3-hydroxy-
Benzenesulfonic acid is called for short TBHB)
3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid (3,5-Dichlorophenolsulfonic acid) of 5-
3, and the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-(3,5-Dichloro-2-hydroxy-benzenesulfonic
Acid is called for short DHBS)
N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt (N-ethyl-N-
(2-hydroxy-3-sulfopropyl)-m-toluidine sodium is called for short TOOS)
Three bromine hydroxy-benzoic acid (Tribromohydroxybenzoic acid)
Two monomethylanilines (Dimethylaniline is called for short DMA)
N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine (N-Ethyl-N-(2-hydroxy-
3-sulfopropyl)-m-toluidine is called for short EHSPT)
2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid) (2,2 '-azino-bis (3-
Ethylbenzthiazoline-6-sulfonic acid) is called for short ABTS
Vanillic acid (Vanillic acid (4-hydroxy-3-
Methoxybenzoic acid) is called for short HMB)
(3-methyl-ethyl-hydroxyaniline is called for short 3-methyl-ethyl-hydroxyanilines
MEHA)
Also above single agent can be made into following pair of agent, more help eliminating inside and outside source phosphoric acid salt (H
2PO
4 -) pollute:
Reagent I
Damping fluid 40--200mmol/l
Pyruvic acid 1--20mmol/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--50000U/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination (one) 0.1--20mmol/l
Reduced form chromogen combination (one) can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.1-20mmol/l or 0.1-20mmol/l is one of in the two.
Reagent II
Damping fluid 40--200mmol/l
Uridine list phosphatase 11--50mmol/l
Stablizer 10--50mmol/l
Reduced form chromogen combination (two) 0.1--20mmol/l
Reduced form chromogen combination (two) can be one of following 14 kinds of compositions of 0.1--20mmol/l: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.
The prescription of two agent is not limited only to above-mentioned prescription, and reduced form chromogen combination () and (two) can transpositions.So can form multiple formulations, not describe in detail one by one at this.
Reagent can also be made into following three reagent, not only more help eliminating inside and outside source phosphoric acid salt (H
2PO
4 -) pollute, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Pyruvic acid 1--20mmol/l
Stablizer 10--50mmol/l
Reduced form chromogen combination (one) 0.1--20mmol/l
Reduced form chromogen combination (one) can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.1-20mmol/l or 0.1-20mmol/l is one of in the two.
Reagent II
Damping fluid 40--200mmol/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Uridine list phosphatase 11--50mmol/l
Stablizer 10--50mmol/l
Reduced form chromogen combination (two) 0.1--20mmol/l
Reduced form chromogen combination (two) can be one of following 14 kinds of compositions of 0.1--20mmol/l: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.
Three doses prescription is not limited only to above-mentioned prescription, and reduced form chromogen combination () and (two) can divide any position that is opened in reagent I, II or III.The composition of reagent I wherein, pyruvic acid can be placed among the reagent II, the composition of reagent II, and pyruvic oxidase, peroxidase etc. also can be placed among the reagent I, so can form multiple formulations, does not describe in detail one by one.
The pH scope of buffer reagent can be 6.0～11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, trolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid, imidazoles (Imidazole) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer 10～80% or 10～50mmol/l among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stablizer can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine or the salt etc.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, the present invention 5 of following system component relation '-the phosphonuclease diagnostic kit is comparatively desirable:
Damping fluid 80--120mmol/l
Inosine list phosphatase 11--5mmol/l
Pyruvic acid 1--10mmol/l
Pyruvic oxidase 5000--20000U/l
Peroxidase 5000--20000U/l
Stablizer 10--50% (cumulative volume)
10--50mmol/l
Reduced form chromogen combination 0.1--10mmol/l
Because the present invention utilizes Enzymology method, enzyme digestion reaction to have the high characteristics of specificity fully, is not vulnerable to the interference of other materials of inside and outside source.Easy, the easy handling of enzyme process method.The specificity of enzyme digestion reaction impels test result accurate.Enzyme digestion reaction all is to carry out under buffer conditions, does not have environmental pollution problem.The enzyme process method does not need special, extra instrument, and testing cost is cheap.Therefore can guarantee to have higher test accuracy, be more convenient for applying.
In addition, one of striking features of the present invention is to eliminate inside and outside source phosphoric acid salt (H
2PO
4 -) pollution, eliminate inside and outside source phosphoric acid salt (H
2PO
4 -) effect occur in the first half of entire reaction time period, at contaminated inside and outside source phosphoric acid salt (H of time second half section
2PO
4 -) be consumed totally, and second half section time test 5 '-the needed phosphoric acid salt (H of activity of 5 '-nucleotidase
2PO
4 -) all be result from 5 '-activity of phosphonuclease.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
5 of present embodiment '-the phosphonuclease diagnostic kit comprises:
Damping fluid 80mmol/l
Uridine list phosphatase 11 mmol/l
Pyruvic acid 1mmol/l
Pyruvic oxidase 5000U/l
Peroxidase 5000U/l
Stablizer 50% (cumulative volume)
The amino anti-arsenic 2mmol/l of 4-
PHENOL 99.8 MIN ((CARBOLIC ACID)) 10mmol/l
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 495～505nm, more than the test commplementary wave length 600nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.
After adding sample and reagent, make it to mix, following reaction take place:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+two
Carbonoxide+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination
PeroxidaseThe indoleamine chromogen or
Quinone-imine chromogen+water
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 495～505nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.
Present embodiment uses 5 '-enzyme linked reactions such as phosphonuclease coupling pyruvic oxidase, peroxidase, with uridine list phosphoric acid is substrate, and reduced form chromogen combined system, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen or indoleamine chromogen, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect 5 '-size of activity of 5 '-nucleotidase.
Embodiment two (two agent)
5 of present embodiment '-the phosphonuclease diagnostic reagent has:
Reagent I
Damping fluid 100mmol/l
Pyruvic acid 5mmol/l
Pyruvic oxidase 12000U/l
Peroxidase 12000U/l
Stablizer 50% (cumulative volume)
The amino anti-arsenic 1mmol/l of 4-
Reagent II
Damping fluid 100mmol/l
Inosine list phosphoric acid 3mmol/l
Stablizer 30mmol/l
2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid 1mmol/l
Measure 5 '-during activity of 5 '-nucleotidase, temperature is controlled at 30 ℃, 15 minutes reaction times, test predominant wavelength 546nm, more than the test commplementary wave length 600nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.
Concrete determination step is:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+two
Carbonoxide+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination
PeroxidaseThe indoleamine chromogen or
Quinone-imine chromogen+water
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 546nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.
The reaction times of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
5 of present embodiment '-the phosphonuclease diagnostic reagent is three doses, has:
Reagent I
Damping fluid 120mmol/l
Pyruvic acid 10mmol/l
Stablizer 50mmol/l
3-methyl-2-Proxel hydrazone 2mmol/l
Reagent II
Damping fluid 120mmol/l
Pyruvic oxidase 20000U/l
Peroxidase 20000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Uridine list phosphoric acid 5mmol/l
Stablizer 50mmol/l
Two monomethylaniline 2mmol/l
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.
Concrete determination step is:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+two
Carbonoxide+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination
PeroxidaseThe indoleamine chromogen or
Quinone-imine chromogen+water
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 578nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.
Embodiment four
5 of present embodiment '-the phosphonuclease diagnostic reagent has:
Reagent I
Damping fluid 120mmol/l
Pyruvic acid 6mmol/l
Pyruvic oxidase 12000U/l
Peroxidase 12000U/l
Stablizer 50% (cumulative volume)
The amino anti-arsenic 1mmol/l of 4-
Reagent II
Damping fluid 120mmol/l
Uridine list phosphoric acid 2mmol/l
Stablizer 20mmol/l
Two monomethylaniline 2mmol/l
Measure 5 '-during activity of 5 '-nucleotidase, temperature is controlled at 30 ℃, 10 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, tested 5 '-volume ratio of phosphonuclease sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.
After adding sample and reagent, make it to mix, following reaction take place:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+two
Carbonoxide+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination
PeroxidaseThe indoleamine chromogen or
Quinone-imine chromogen+water
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 578nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.
The reaction times of each reactions steps was controlled at 5 minutes.
In a word, experiment showed, and adopt measuring method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, tolerance range good, is not subjected to the pollution of inside and outside source material.
Claims (10)
1. one kind 5 '-the activity of 5 '-nucleotidase measuring method, step is as follows:
1), with sample and the reagent mix of mainly forming by uridine list phosphoric acid, pyruvic acid, pyruvic oxidase, peroxidase, make it to take place following reaction:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+two
Carbonoxide+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination
PeroxidaseThe indoleamine chromogen or
Quinone-imine chromogen+water
2), detect the end reaction thing in the speed that predominant wavelength 400--600nm absorbancy rises, calculate 5 '-the active size of phosphonuclease.
According to claim 1 described 5 '-the activity of 5 '-nucleotidase measuring method, it is characterized in that: described step 2) be, the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that the 400--600nm absorbancy rises, calculate 5 '-the active size of phosphonuclease.
3, according to claim 1 or 2 described mensuration 5 '-method of activity of 5 '-nucleotidase, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction times was controlled at 2-30 minute.
4, according to the described mensuration 5 of claim 1 '-method of activity of 5 '-nucleotidase, it is characterized in that: tested 5 '-ratio control of phosphonuclease sample and reagent is 1/10 to 1/500.
5. one kind 5 '-the phosphonuclease diagnostic kit, be grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Inosine list phosphatase 11--50mmol/l
Pyruvic acid 1--20mmol/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--50000U/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination 0.1--20mmol/l
According to claim 5 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: described reagent is made into single agent, two agent or three doses.
According to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, ammonium sulfate or the salt.
According to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: the pH scope of described buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.
9, according to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: one of following 14 kinds of compositions of 3-methyl-2-Proxel hydrazone that described reduced form chromogen is 0.1-20mmol/l and 0.1-20mmol/l combine: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.
10, according to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: described reduced form chromogen is combined as by the amino anti-arsenic of the 4-of 0.1-20mmol/l and one of 14 kinds of compositions below the 0.1-20mmol/l and combines: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.
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|---|---|---|---|
| CN 200410064911 CN1760368A (en) | 2004-10-11 | 2004-10-11 | Method for testing activity of 5'-nucleotidase, diagnosis kit for 5'-nucleotidase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410064911 CN1760368A (en) | 2004-10-11 | 2004-10-11 | Method for testing activity of 5'-nucleotidase, diagnosis kit for 5'-nucleotidase |
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|---|---|
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