CN1769470A - Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit - Google Patents
Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit Download PDFInfo
- Publication number
- CN1769470A CN1769470A CN 200410065603 CN200410065603A CN1769470A CN 1769470 A CN1769470 A CN 1769470A CN 200410065603 CN200410065603 CN 200410065603 CN 200410065603 A CN200410065603 A CN 200410065603A CN 1769470 A CN1769470 A CN 1769470A
- Authority
- CN
- China
- Prior art keywords
- adenosine
- adenosine deaminase
- reagent
- glutamic acid
- enzyme
- 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
- 238000000034 method Methods 0.000 title claims abstract description 35
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 title claims description 56
- 102000055025 Adenosine deaminases Human genes 0.000 title claims description 56
- 230000000694 effects Effects 0.000 title claims description 23
- 238000003745 diagnosis Methods 0.000 title claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 77
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims abstract description 68
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 55
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims abstract description 40
- 229960005305 adenosine Drugs 0.000 claims abstract description 34
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000005515 coenzyme Substances 0.000 claims description 47
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 229960002989 glutamic acid Drugs 0.000 claims description 27
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 27
- 238000013016 damping Methods 0.000 claims description 26
- 239000012530 fluid Substances 0.000 claims description 26
- 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 claims description 25
- 108020000311 Glutamate Synthase Proteins 0.000 claims description 23
- -1 2-oxoglutaric acid ester Chemical class 0.000 claims description 21
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims description 20
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 20
- 102000005396 glutamine synthetase Human genes 0.000 claims description 17
- 108020002326 glutamine synthetase Proteins 0.000 claims description 17
- 229910021529 ammonia Inorganic materials 0.000 claims description 15
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- 230000001186 cumulative effect Effects 0.000 claims description 9
- 230000035484 reaction time Effects 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- 239000001177 diphosphate Substances 0.000 claims description 6
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 6
- 235000011180 diphosphates Nutrition 0.000 claims description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 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
- 229960003966 nicotinamide Drugs 0.000 claims description 2
- 235000005152 nicotinamide Nutrition 0.000 claims description 2
- 239000011570 nicotinamide Substances 0.000 claims description 2
- 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 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
- JRIZSBGDMDSKRF-DEGSGYPDSA-N [(2s,3s,4s,5s)-5-(6-aminopurin-9-yl)-3,4-diphosphonooxyoxolan-2-yl]methyl dihydrogen phosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1O[C@@H](COP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O JRIZSBGDMDSKRF-DEGSGYPDSA-N 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
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 1
- 229940043257 glycylglycine Drugs 0.000 claims 1
- 239000002953 phosphate buffered saline Substances 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 12
- 108090000790 Enzymes Proteins 0.000 abstract description 12
- 230000009615 deamination Effects 0.000 abstract 3
- 238000006481 deamination reaction Methods 0.000 abstract 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 abstract 2
- 235000013922 glutamic acid Nutrition 0.000 abstract 2
- 239000004220 glutamic acid Substances 0.000 abstract 2
- 239000002253 acid Substances 0.000 abstract 1
- 239000000376 reactant Substances 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 abstract 1
- 239000003381 stabilizer Substances 0.000 abstract 1
- 239000001226 triphosphate Substances 0.000 abstract 1
- 235000011178 triphosphate Nutrition 0.000 abstract 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 17
- 230000008878 coupling Effects 0.000 description 7
- 238000010168 coupling process Methods 0.000 description 7
- 238000005859 coupling reaction Methods 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229930010555 Inosine Natural products 0.000 description 2
- 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 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 2
- 238000006491 synthase reaction Methods 0.000 description 2
- 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
- 206010003445 Ascites Diseases 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 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
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- 108010044467 Isoenzymes Proteins 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
- 230000001154 acute effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 210000004910 pleural fluid Anatomy 0.000 description 1
- QVLTXCYWHPZMCA-UHFFFAOYSA-N po4-po4 Chemical compound OP(O)(O)=O.OP(O)(O)=O QVLTXCYWHPZMCA-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention relates to a method for measuring the active of the adenosine deamination enzyme and the digonose reagent box for adenosine deamination enzyme, which belongs to the physic checking measuring technique area. This reagent box includes cushion solution, adenosine, glutamic acid, 2-2-ketoglutaric acid,reducing assist enzyme,adenylic triphosphate acid, glutamine synthetic enzyme, glutamic acid synthetic enzyme, stabilizing agent, this method mixes the smple with the reagent by a certain volum proportion to make it fall into the enzyme couplet action, then puts the finally reactant under the biochemistry analyzer to check the absorbenfy changing situation of the main wave to work out the active of the adenosine deamination enzyme. This invention can achive the needed result completely through the biochemistry analyzer, the sensitivity and the definition is high, and it doesn't suffer the pollution from the environment.
Description
Technical field
The present invention relates to a kind of method of measuring activity of adenosine deaminase, the invention still further relates to simultaneously, belong to medical test determination techniques field in order to realize the adenosine deaminase diagnosis reagent kit of this method.
Background technology
Medical research shows, adenosine deaminase is the nucleic acid metabolism enzyme that a kind of and body cell immunocompetence have important relationship.Therefore, the mensuration of activity of adenosine deaminase is used as good pernicious ascites pleural fluid, cerebrospinal fluid differential diagnosis, severe combined immunodeficiency disease (SCID), acute and chronic hepatitis, liver cirrhosis, the important diagnostic sign that diseases such as liver cancer are differentiated.
The activity determination method of adenosine deaminase mainly contains active nucleus method, physical method and biochemical process.Understand according to the applicant, generally adopt the UV-light method at present in the world, its measuring principle is: adenosine (white crystalline powder)+water (H2O)
Adenosine deaminaseInosine (Inosine)+ammonium ion (NH4+), the inosine that this reflection process generates can be that the 265nm place manifests at wavelength, therefore can pass through the size of the big or small directly reflection activity of adenosine deaminase of this wavelength place absorbancy of mensuration.Yet this method can't be measured with the visible light analysis instrument of general hospital, and needs to use special ultraviolet light analyzer, therefore is difficult to apply conscientiously.
Retrieval finds, application number is 03128092.7, the applying date is that the Chinese patent application that 2003.05.29, name are called " a kind of reagent and method for making thereof of measuring adenosine deaminase " discloses the mensuration reagent of using the preparation of enzyme reaction product oxidized form nicotinoyl ammonia coenzyme.The enzymic measuring reagent of this invention indication does not add assaying reaction required reduced form nicotinoyl ammonia coenzyme or its analogue, and add its reaction product oxidized form nicotinoyl ammonia coenzyme or its analogue, and generate required enzyme of reduced coenzyme and substrate system accordingly, by in reagent, forming long response time systems such as enzyme-substrate-NAD or enzyme-substrate-NADP, this class oxidized coenzyme or the slow circulation of its analogue are reduced to reduced form nicotinoyl ammonia coenzyme or its analogue, when the reduced form nicotinoyl ammonia coenzyme that is reduced or its analogue reached certain concentration, the enzyme process reagent of this invention indication just can reach the purpose of measuring adenosine deaminase in the sample and isozymes activity thereof.This characteristic feature of an invention is to provide an endogenous synthesizing adenosine desaminase to react the adenosine deaminase reagent of needed reduced form nicotinoyl ammonia coenzyme for the test of adenosine deaminase.One of its shortcoming is, this system is in the needed reduced form nicotinoyl of formation reaction ammonia coenzyme, also offset the activity of part adenosine deaminase reagent (reaction of adenosine deaminase coupling glutamate dehydrogenase must be oxidized to reduced form nicotinoyl ammonia coenzyme oxidized form nicotinoyl ammonia coenzyme), caused the accuracy of reagent test to reduce greatly.Two of its shortcoming is that this reagent must react for some time before official testing in advance, so that can produce enough reduced form nicotinoyl ammonia coenzyme, has so just caused the result that slow action cannot save a critical situation, brings very big inconvenience to test.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of measuring method that can overcome the activity of adenosine deaminase of above prior art shortcoming, provide simultaneously in order to realize the adenosine deaminase diagnosis reagent kit of this method.Adopt the reagent in this test kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out adenosine deaminase activity determination, and finding speed is fast, accuracy is high, thereby can obtains practical applying.
It is as follows that the present invention measures the method steps of activity of adenosine deaminase:
1), with sample and the reagent mix of mainly forming by adenosine, L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate, glutamine synthetase, NADPH-linked glutamate synthase, make it to take place the reaction of following method principle:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic acid+oxidized coenzyme
2), detect the end reaction thing and detecting the speed that predominant wavelength 340nm absorbancy descends, calculate the active size of adenosine deaminase.
Common 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 340nm absorbancy descends, draw the adenosine deaminase measurement result.
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.
This method is used adenosine deaminase coupling glutamine synthetase, NADPH-linked glutamate synthase reaction continuous monitoring method.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling glutamine synthetase again, generate glutamine with adenosine triphosphate and L-glutamic acid, glutamine is the effect by the coupling NADPH-linked glutamate synthase again, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place), thereby measured the speed that reduced coenzyme descends in 340nm place absorbancy, by measuring the speed that 340nm place absorbancy descends, can calculate the active size of adenosine deaminase.
Adenosine deaminase diagnosis reagent kit in order to realization the inventive method can be single agent, comprising:
Damping fluid 40--200mmol/1
Adenosine 0.5--50mmol/l
L-glutamic acid 1--30mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
Glutamine synthetase 1000--50000U/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Also above single agent can be made into following pair of agent, more help eliminating inside and outside source ammonia pollution:
Reagent I
Damping fluid 40--200mmol/l
L-glutamic acid 1--30mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
Glutamine synthetase 1000--50000U/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Stablizer 10--50mmol/l
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, and L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate, glutamine synthetase, NADPH-linked glutamate synthase etc. can be placed on reagent II.Reagent II composition wherein, adenosine also can be placed on reagent I, so can form multiple formulations, does 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 ammonia pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
L-glutamic acid 1--30mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
Stablizer 10--50mmol/l
Reagent II
Damping fluid 40--200mmol/l
Glutamine synthetase 1000--50000U/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Stablizer 10--50mmol/l
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, and L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, glutamine synthetase, NADPH-linked glutamate synthase etc. also can be placed among reagent I or the reagent III.Reagent III composition wherein, adenosine also can be placed among reagent I or the reagent II, so can form multiple formulations, do not describe in detail one by one at this.
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, phosphoric acid salt (Phosphate) 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.
Above reduced coenzyme can be reduced form nicotinamide coenzyme or derivatives thereofs such as NADPH, NADH or thio-NADH.
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, and the adenosine deaminase diagnosis reagent kit of the present invention of following system component relation is comparatively desirable:
Damping fluid 80--120mmol/l
Adenosine 2--10mmol/l
L-glutamic acid 5--10mmol/l
2-oxoglutaric acid ester 5--20mmol/l
Reduced coenzyme 0.2--0.3mmol/l
Adenosine triphosphate 2--8mmol/l
Glutamine synthetase 4000--10000U/l
NADPH-linked glutamate synthase 4000--10000U/l
Stablizer 20--50% (cumulative volume)
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 the pollution that can eliminate inside and outside source ammonia, the effect of eliminating inside and outside source ammonia occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source ammonia, and all be the activity that results from adenosine deaminase at the needed ammonia of second half section time test activity of adenosine deaminase.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
The adenosine deaminase diagnosis reagent kit of present embodiment comprises:
Damping fluid 80mmol/l
Adenosine 2mmol/l
L-glutamic acid 5mmol/l
2-oxoglutaric acid ester 5mmol/l
Reduced coenzyme 0.2mmo/l
Adenosine triphosphate 2mmol/l
Glutamine synthetase 4000U/l
NADPH-linked glutamate synthase 4000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, and the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy descends, thereby calculate the active size of adenosine deaminase.
Present embodiment is used adenosine deaminase coupling glutamine synthetase, NADPH-linked glutamate synthase reaction continuous monitoring method.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling glutamine synthetase again, generate glutamine with adenosine triphosphate and L-glutamic acid, glutamine is the effect by the coupling NADPH-linked glutamate synthase again, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place), thereby measured the speed that reduced coenzyme descends in 340nm place absorbancy, by measuring the speed that 340nm place absorbancy descends, can calculate the active size of adenosine deaminase.
Embodiment two (two agent)
The adenosine deaminase diagnosing reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
L-glutamic acid 8mmol/l
2-oxoglutaric acid ester 12mmol/l
Reduced coenzyme 0.25mmo/l
Adenosine triphosphate 5mmol/l
Glutamine synthetase 7000U/l
NADPH-linked glutamate synthase 7000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine 6mmol/l
Stablizer 20mmol/l
When measuring activity of adenosine deaminase, temperature is controlled at 30 ℃, 15 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
Concrete determination step is:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy descends, thereby calculate the active size of adenosine deaminase.
The reaction times of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
The adenosine deaminase diagnosing reagent of present embodiment is three doses, has:
Reagent I
Damping fluid 120mmol/l
L-glutamic acid 10mmol/l
2-oxoglutaric acid ester 20mmol/l
Reduced coenzyme 0.3mmo/l
Adenosine triphosphate 8mmol/l
Stablizer 20mmol/l
Reagent II
Damping fluid 120mmol/l
Glutamine synthetase 10000U/l
NADPH-linked glutamate synthase 10000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Adenosine 10mmol/l
Stablizer 20mmol/l
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, and the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
Concrete determination step is:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy descends, thereby calculate the active size of adenosine deaminase.
The reaction times of each reactions steps was controlled at 20 minutes.
Embodiment four
The adenosine deaminase diagnosing reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
L-glutamic acid 5mmol/l
2-oxoglutaric acid ester 12mmol/l
Reduced coenzyme 0.3mmo/l
Adenosine triphosphate 2mmol/l
Stablizer 20mmol/l
Reagent II
Damping fluid 100mmol/l
Adenosine 2mmol/l
Glutamine synthetase 10000U/l
NADPH-linked glutamate synthase 10000U/l
Stablizer 50% (cumulative volume)
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, and the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic acid+oxidized coenzyme
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy descends, thereby calculate the active size of adenosine deaminase.
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 (9)
1. method of measuring activity of adenosine deaminase, step is as follows:
1), with sample and the reagent mix of mainly forming by adenosine, L-glutamic acid, 2-oxoglutaric acid ester, reduced coenzyme, adenosine triphosphate, glutamine synthetase, NADPH-linked glutamate synthase, make it to take place the reaction of following method principle:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+
Phosphate radical+glutamine
Glutamine+2-oxoglutaric acid ester+reduced coenzyme
NADPH-linked glutamate synthase
L-glutamic acid+oxidized coenzyme
2), detect the end reaction thing and detecting the speed that predominant wavelength 340nm absorbancy descends, calculate the active size of adenosine deaminase.
2. according to the method for the described mensuration activity of adenosine deaminase of claim 1, 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, detection is calculated the active size of adenosine deaminase in the speed that predominant wavelength 340nm absorbancy descends.
3, according to the method for claim 1 or 2 described mensuration activity of adenosine deaminase, 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 method for claim 1 or 2 described mensuration activity of adenosine deaminase, it is characterized in that: the ratio control of sample and reagent is 1/10 to 1/250.
5. adenosine deaminase diagnosis reagent kit is grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
L-glutamic acid 1--30mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Adenosine triphosphate 1--10mmol/l
Glutamine synthetase 1000--50000U/l
NADPH-linked glutamate synthase 1000--50000U/l
Stablizer 10--80% (cumulative volume)
6. according to the described adenosine deaminase diagnosis reagent kit of claim 5, it is characterized in that: described reagent is made into single agent, two agent or three doses.
7. according to claim 5 or 6 described adenosine deaminase diagnosis reagent kits, it is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, ammonium sulfate or the salt.
8. according to claim 5 or 6 described adenosine deaminase diagnosis reagent kits, 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, phosphate buffered saline 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 adenosine deaminase diagnosis reagent kits, it is characterized in that: described reduced coenzyme is a kind of in NADPH, NADH or the thio-NADH reduced form nicotinamide coenzyme or derivatives thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410065603 CN1769470A (en) | 2004-11-05 | 2004-11-05 | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410065603 CN1769470A (en) | 2004-11-05 | 2004-11-05 | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1769470A true CN1769470A (en) | 2006-05-10 |
Family
ID=36751014
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410065603 Pending CN1769470A (en) | 2004-11-05 | 2004-11-05 | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1769470A (en) |
-
2004
- 2004-11-05 CN CN 200410065603 patent/CN1769470A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1769481A (en) | Blood ammonia content determination method and blood ammonia diagnosis kit | |
| CN1749756A (en) | Method for detecting blood ammonia content and blood ammonia diagnostic reagent kit | |
| CN1778963A (en) | Determination of blood ammonia content and blood ammonia diagnostic reagent kit | |
| CN1746316A (en) | Determination of adenosine deaminase activity and diagnostic kit of adenosine deaminase | |
| CN1769470A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1778944A (en) | Determination of creatinine content and creatinine diagnostic reagent kit | |
| CN1769483A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1778947A (en) | Creatinine content determination and creatinine diagnostic reagent kit | |
| CN1769484A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1769478A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1302119C (en) | Method for measuring 5'-nucleotidase activity and diagnostic reagent kit of 5'-nucleotidase | |
| CN1757754A (en) | Determination method of creatnine content and reagent box for diagnosing creatnine | |
| CN1769482A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1766638A (en) | Creatinine content determination method and creatinine diagnosis kit | |
| CN1769477A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1766640A (en) | Creatinine content determination method and creatinine diagnosis kit | |
| CN1769473A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1757748A (en) | Determination method of creatnine content and creatnine diagnosis reagent box | |
| CN1769479A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1769475A (en) | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit | |
| CN1778946A (en) | Determination of blood ammonia content and blood ammonia diagnostic reagent kit | |
| CN1778937A (en) | Determination of blood ammonia content and blood ammonia diagnostic reagent kit | |
| CN1749411A (en) | Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase | |
| CN1757750A (en) | Method of determining creatnine content and reagent box for diagnosing creatnine | |
| CN1758050A (en) | Determination method of creatinine content and reagent box for diagnosing creatinine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |