CN1769477A - Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit - Google Patents
Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit Download PDFInfo
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- CN1769477A CN1769477A CN 200410065601 CN200410065601A CN1769477A CN 1769477 A CN1769477 A CN 1769477A CN 200410065601 CN200410065601 CN 200410065601 CN 200410065601 A CN200410065601 A CN 200410065601A CN 1769477 A CN1769477 A CN 1769477A
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- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 title claims abstract description 59
- 102000055025 Adenosine deaminases Human genes 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 33
- 230000000694 effects Effects 0.000 title claims abstract description 27
- 238000003745 diagnosis Methods 0.000 title claims abstract description 13
- 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 62
- 239000005515 coenzyme Substances 0.000 claims abstract description 62
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- 239000002126 C01EB10 - Adenosine Substances 0.000 claims abstract description 31
- 229960005305 adenosine Drugs 0.000 claims abstract description 31
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims abstract description 25
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims abstract description 25
- 102000013460 Malate Dehydrogenase Human genes 0.000 claims abstract description 24
- 108010026217 Malate Dehydrogenase Proteins 0.000 claims abstract description 24
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 46
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims description 46
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 40
- 238000006243 chemical reaction Methods 0.000 claims description 28
- 238000013016 damping Methods 0.000 claims description 26
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- 150000002085 enols Chemical class 0.000 claims description 23
- 229940107700 pyruvic acid Drugs 0.000 claims description 23
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- -1 2-oxoglutaric acid ester Chemical class 0.000 claims description 21
- 101710088194 Dehydrogenase Proteins 0.000 claims description 21
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 21
- 239000001630 malic acid Substances 0.000 claims description 21
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- 235000011089 carbon dioxide Nutrition 0.000 claims description 20
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- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 13
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 13
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- 229960004418 trolamine Drugs 0.000 claims description 3
- 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 2
- 150000004676 glycans Chemical class 0.000 claims description 2
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- 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
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- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims 2
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- UCWIJRLALQMQMP-UHFFFAOYSA-N 2-aminobutanoic acid;4-aminobutanoic acid Chemical compound CCC(N)C(O)=O.NCCCC(O)=O UCWIJRLALQMQMP-UHFFFAOYSA-N 0.000 claims 1
- PDLNHDSYGLTYDS-UHFFFAOYSA-N 3-aminopropanoic acid;hydrochloride Chemical compound Cl.NCCC(O)=O PDLNHDSYGLTYDS-UHFFFAOYSA-N 0.000 claims 1
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- 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
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- 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
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- 102000008214 Glutamate decarboxylase Human genes 0.000 abstract 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for determining the activity of adenosine deaminase, and also the reagent kit for adenosine deaminase diagnosis. The reagent kit comprises cushioning solution, adenosine, 2-ketoglutaric ester, deacidized type coenzyme, phosphoenolpyruvate phosphatase, glutamate dehydrogenase, glutamic acid decarboxylase, phosphoenolpyruvate pyruvate carboxylase, malic dehydrogenase, and stabilizer. By mixing sample and reagent of a predetermiend volumetric ratio, generating coupling reaction between them, subjecting the final reactant to biochemiscal analyser, the main wavelength absorbancy variance ratio (speed) can be detected, and the activity of the adenosine deaminase can thus be measured. The method of the invention can be used to obtain the needed measurement result purely through biochemical analytic instruments, and advantages of the method include higher sensibility, better accuracy, less susceptibility to contamination of internal or external materials, and easy application.
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, 2-oxoglutaric acid ester, reduced coenzyme, phosphoenolpyruvic acid, glutamate dehydrogenase, L-Glutamic decarboxylase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase (malic acid dehydrogenase), make it to take place the reaction of following principle:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenase
L-glutamic acid+coenzyme+water
L-glutamic acid
L-Glutamic decarboxylase4-aminobutyric acid (aminobutanoate)+carbonic acid gas
Carbonic acid gas+phosphoenolpyruvic acid
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malate dehydrogenase (malic acid dehydrogenase)Oxysuccinic acid+oxidized coenzyme
2), detect the end reaction thing in 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 glutamate dehydrogenase, L-Glutamic decarboxylase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase enzyme reaction continuous monitoring method.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling glutamate dehydrogenase again, produce L-glutamic acid, produce carbonic acid gas by L-Glutamic decarboxylase catalysis, carbonic acid gas produces oxaloacetic acid with phosphoenolpyruvic acid again under the effect of phosphoric acid enol pyruvic acid carboxylase, effect by the coupling malate dehydrogenase (malic acid dehydrogenase) 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.Advantage of the present invention is that reaction has utilized the reduced coenzyme of two molecules, has also produced the oxidized coenzyme of two molecules, so absorbancy has the duple pace of change, and sensitivity has also just improved two times.
Adenosine deaminase diagnosis reagent kit in order to realization the inventive method can be single agent, comprising:
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Phosphoenolpyruvic acid 1--10mmol/l
Glutamate dehydrogenase 1000--50000U/l
L-Glutamic decarboxylase 1000--50000U/l
Phosphoric acid enol pyruvic acid carboxylase 500--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Also above single agent following pair of agent be can be made into, inside and outside source ammonia, L-glutamic acid, carbon dioxide pollution more helped eliminating:
Reagent I
Damping fluid 40--200mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Phosphoenolpyruvic acid 1--10mmol/l
Glutamate dehydrogenase 1000--50000U/l
L-Glutamic decarboxylase 1000--50000U/l
Phosphoric acid enol pyruvic acid carboxylase 500--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Stablizer 10--80% (cumulative volume)
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, 2-oxoglutaric acid ester, reduced coenzyme, phosphoenolpyruvic acid, glutamate dehydrogenase, L-Glutamic decarboxylase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase (malic acid dehydrogenase) 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, L-glutamic acid, carbon dioxide pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Phosphoenolpyruvic acid 1--10mmol/l
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Glutamate dehydrogenase 1000--50000U/l
L-Glutamic decarboxylase 1000--50000U/l
Phosphoric acid enol pyruvic acid carboxylase 500--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 1000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Stablizer 10--80% (cumulative volume)
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, and 2-oxoglutaric acid ester, reduced coenzyme, phosphoenolpyruvic acid etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, glutamate dehydrogenase, L-Glutamic decarboxylase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase (malic acid dehydrogenase) 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 3--20mmol/l
2-oxoglutaric acid ester 6--30mmol/l
Reduced coenzyme 0.2--0.3mmo/l
Phosphoenolpyruvic acid 2--8mmol/l
Glutamate dehydrogenase 6000--10000U/l
L-Glutamic decarboxylase 6000--10000U/l
Phosphoric acid enol pyruvic acid carboxylase 2000--8000U/l
Malate dehydrogenase (malic acid dehydrogenase) 6000--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, L-glutamic acid, carbonic acid gas, the effect of eliminating inside and outside source ammonia, L-glutamic acid, carbonic acid gas occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source ammonia, L-glutamic acid, carbonic acid gas, and all be the activity that results from adenosine deaminase at the needed ammonia of second half section time test activity of adenosine deaminase, L-glutamic acid, carbonic acid gas.
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 3mmol/l
2-oxoglutaric acid ester 6mmol/l
Reduced coenzyme 0.2mmo/l
Phosphoenolpyruvic acid 2mmol/l
Glutamate dehydrogenase 6000U/l
L-Glutamic decarboxylase 6000U/l
Phosphoric acid enol pyruvic acid carboxylase 2000U/l
Malate dehydrogenase (malic acid dehydrogenase) 6000U/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
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenase
L-glutamic acid+coenzyme+water
L-glutamic acid
L-Glutamic decarboxylase4-aminobutyric acid (aminobutanoate)+carbonic acid gas
Carbonic acid gas+phosphoenolpyruvic acid
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malate dehydrogenase (malic acid dehydrogenase)Oxysuccinic 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 glutamate dehydrogenase, L-Glutamic decarboxylase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase enzyme reaction continuous monitoring method.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling glutamate dehydrogenase again, produce L-glutamic acid, produce carbonic acid gas by L-Glutamic decarboxylase catalysis, carbonic acid gas produces oxaloacetic acid with phosphoenolpyruvic acid again under the effect of phosphoric acid enol pyruvic acid carboxylase, effect by the coupling malate dehydrogenase (malic acid dehydrogenase) 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
2-oxoglutaric acid ester 18mmol/l
Reduced coenzyme 0.25mmo/l
Phosphoenolpyruvic acid 5mmol/l
Glutamate dehydrogenase 8000U/l
L-Glutamic decarboxylase 8000U/l
Phosphoric acid enol pyruvic acid carboxylase 5000U/l
Malate dehydrogenase (malic acid dehydrogenase) 8000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine 12mmol/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
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenase
L-glutamic acid+coenzyme+water
L-glutamic acid
L-Glutamic decarboxylase4-aminobutyric acid (aminobutanoate)+carbonic acid gas
Carbonic acid gas+phosphoenolpyruvic acid
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malate dehydrogenase (malic acid dehydrogenase)Oxysuccinic 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
2-oxoglutaric acid ester 30mmol/l
Reduced coenzyme 0.3mmo/l
Phosphoenolpyruvic acid 8mmol/l
Stablizer 20mmol/l
Reagent II
Damping fluid 120mmol/l
Glutamate dehydrogenase 10000U/l
L-Glutamic decarboxylase 10000U/l
Phosphoric acid enol pyruvic acid carboxylase 8000U/l
Malate dehydrogenase (malic acid dehydrogenase) 10000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Adenosine 20mmol/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
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenase
L-glutamic acid+coenzyme+water
L-glutamic acid
L-Glutamic decarboxylase4-aminobutyric acid (aminobutanoate)+carbonic acid gas
Carbonic acid gas+phosphoenolpyruvic acid
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malate dehydrogenase (malic acid dehydrogenase)Oxysuccinic 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
Glutamate dehydrogenase 6000U/l
L-Glutamic decarboxylase 6000U/l
Phosphoric acid enol pyruvic acid carboxylase 8000U/l
Malate dehydrogenase (malic acid dehydrogenase) 6000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine 5mmol/l
2-oxoglutaric acid ester 10mmol/l
Reduced coenzyme 0.3mmo/l
Phosphoenolpyruvic acid 3mmol/l
Stablizer 20mmol/l
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
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenase
L-glutamic acid+coenzyme+water
L-glutamic acid
L-Glutamic decarboxylase4-aminobutyric acid (aminobutanoate)+carbonic acid gas
Carbonic acid gas+phosphoenolpyruvic acid
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malate dehydrogenase (malic acid dehydrogenase)Oxysuccinic 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, 2-oxoglutaric acid ester, reduced coenzyme, phosphoenolpyruvic acid, glutamate dehydrogenase, L-Glutamic decarboxylase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase (malic acid dehydrogenase), make it to take place the reaction of following principle:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenase
L-glutamic acid+coenzyme+water
L-glutamic acid
L-Glutamic decarboxylase4-aminobutyric acid (aminobutanoate)
+ carbonic acid gas
Carbonic acid gas+phosphoenolpyruvic acid
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malate dehydrogenase (malic acid dehydrogenase)Oxysuccinic acid+oxidized coenzyme
2), detect the end reaction thing in 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, detect the speed that predominant wavelength 340nm absorbancy descends, calculate the active size of adenosine deaminase.
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
2-oxoglutaric acid ester 1--50mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Phosphoenolpyruvic acid 1--10mmol/l
Glutamate dehydrogenase 1000--50000U/l
L-Glutamic decarboxylase 1000--50000U/l
Phosphoric acid enol pyruvic acid carboxylase 500--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 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.
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| CN 200410065601 CN1769477A (en) | 2004-11-05 | 2004-11-05 | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit |
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|---|---|---|---|
| CN 200410065601 CN1769477A (en) | 2004-11-05 | 2004-11-05 | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit |
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