CN1760373A - Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase - Google Patents
Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase Download PDFInfo
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- CN1760373A CN1760373A CN 200410064913 CN200410064913A CN1760373A CN 1760373 A CN1760373 A CN 1760373A CN 200410064913 CN200410064913 CN 200410064913 CN 200410064913 A CN200410064913 A CN 200410064913A CN 1760373 A CN1760373 A CN 1760373A
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- phosphonuclease
- pyruvic
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Abstract
A reagent kit for diagnosing 5'-nucleotidase is composed of buffer liquid, uridine monophosphate, pyruvic acid, phosphoenolpyruvic acid, reduced coenzyme, phosphoneolphruvate carboxylase, malate dehydrogenase and stabilizer. 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, phosphoenolpyruvic acid, reduced coenzyme, pyruvic oxidase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase (malic acid dehydrogenase), make it to take place the reaction of following principle:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+
Carbonic acid gas+hydrogen peroxide
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 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 340nm absorbancy descends, 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 '-phosphonuclease coupling pyruvic oxidase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase enzyme reaction continuous monitoring method.5 '-phosphonuclease enzymolysis uridine list phosphoric acid generation monophosphate, pyruvic oxidase and phosphoric acid enol pyruvic acid carboxylase remake with the last oxaloacetic acid that produces then, and by the effect of coupling malate dehydrogenase (malic acid dehydrogenase), oxidation NAD (P) H becomes NAD (P) again
+Thereby, measured the speed that NAD (P) H descends in 340nm place absorbancy, by measuring the speed that 340nm place absorbancy descends, can calculate 5 '-phosphonuclease active big or small.Though monophosphate is repeated to use in system, that final speed of reaction still depends on is initial 5 '-the initial monophosphate concentration that size produced of activity of 5 '-nucleotidase determines.
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--10mmol/l
Pyruvic acid 1--10mmol/l
Phosphoenolpyruvic acid 1--20mmol/l
Reduced coenzyme 0.2--0.3mmol/l
Pyruvic oxidase 2000--20000U/l
Phosphoric acid enol pyruvic acid carboxylase 2000--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 2000--20000U/l
Stablizer 10--80% (cumulative volume)
Also above single agent can be made into following pair of agent:
Reagent I
Damping fluid 40--200mmol/l
Pyruvic acid 1--10mmol/l
Phosphoenolpyruvic acid 1--20mmol/l
Reduced coenzyme 0.2--0.3mmol/l
Pyruvic oxidase 2000--20000U/l
Phosphoric acid enol pyruvic acid carboxylase 2000--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 2000--20000U/l
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Uridine list phosphatase 11--10mmol/l
Stablizer 10--50mmol/l
Reagent can also be made into following three reagent, more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Pyruvic acid 1--10mmol/l
Phosphoenolpyruvic acid 1--20mmol/l
Reduced coenzyme 0.2--0.3mmol/l
Stablizer 10--50mmol/l
Reagent II
Damping fluid 40--200mmol/l
Pyruvic oxidase 2000--20000U/l
Phosphoric acid enol pyruvic acid carboxylase 2000--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 2000--20000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Uridine list phosphatase 11--10mmol/l
Stablizer 10--50mmol/l
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, pyruvic acid, phosphoenolpyruvic acid, reduced coenzyme etc. can be placed among the reagent II, reagent II composition wherein, pyruvic oxidase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase (malic acid dehydrogenase) etc. also can be placed among the reagent I, so can form multiple formulations, 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.
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, the present invention 5 of following system component relation '-the phosphonuclease diagnostic kit is comparatively desirable:
Damping fluid 80--120mmol/l
Uridine list phosphatase 11--5mmol/l
Pyruvic acid 1--10mmol/l
Phosphoenolpyruvic acid 1--8mmol/l
Reduced coenzyme 0.2--0.3mmol/l
Pyruvic oxidase 6000--20000U/l
Phosphoric acid enol pyruvic acid carboxylase 6000--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 6000--20000U/l
Stablizer 10--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.
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
Phosphoenolpyruvic acid 1mmol/l
Reduced coenzyme 0.2mmol/l
Pyruvic oxidase 6000U/l
Phosphoric acid enol pyruvic acid carboxylase 6000U/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, 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, theoretical k value-4180.
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+
Carbonic acid gas+hydrogen peroxide
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 5 '-the active size of phosphonuclease.
Present embodiment uses 5 '-phosphonuclease coupling pyruvic oxidase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase enzyme reaction continuous monitoring method.5 '-phosphonuclease enzymolysis uridine list phosphoric acid generation monophosphate, pyruvic oxidase and phosphoric acid enol pyruvic acid carboxylase remake with the last oxaloacetic acid that produces then, and by the effect of coupling malate dehydrogenase (malic acid dehydrogenase), oxidation NAD (P) H becomes NAD (P) again
+Thereby, measured the speed that NAD (P) H descends in 340nm place absorbancy, by measuring the speed that 340nm place absorbancy descends, can calculate 5 '-phosphonuclease active big or small.Though monophosphate is repeated to use in system, that final speed of reaction still depends on is initial 5 '-the initial monophosphate concentration that size produced of activity of 5 '-nucleotidase determines.
Embodiment two (two agent)
5 of present embodiment '-the phosphonuclease diagnostic reagent has:
Reagent I
Damping fluid 100mmol/l
Pyruvic acid 6mmol/l
Phosphoenolpyruvic acid 5mmol/l
Reduced coenzyme 0.25mmol/l
Pyruvic oxidase 13000U/l
Phosphoric acid enol pyruvic acid carboxylase 13000U/l
Malate dehydrogenase (malic acid dehydrogenase) 13000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Uridine list phosphoric acid 3mmol/l
Stablizer 30mmol/l
Measure 5 '-during activity of 5 '-nucleotidase, temperature is controlled at 30 ℃, 15 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, tested 5 '-volume ratio of phosphonuclease 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:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+
Carbonic acid gas+hydrogen peroxide
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 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
Phosphoenolpyruvic acid 8mmol/l
Reduced coenzyme 0.3mmol/l
Stablizer 50mmol/l
Reagent II
Damping fluid 120mmol/l
Pyruvic oxidase 20000U/l
Phosphoric acid enol pyruvic acid carboxylase 20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 20000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Uridine list phosphoric acid 5mmol/l
Stablizer 50mmol/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, tested 5 '-volume ratio of phosphonuclease 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:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+
Carbonic acid gas+hydrogen peroxide
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 5 '-the active size of phosphonuclease.
The reaction times of each reactions steps was controlled at 20 minutes.
Embodiment four
5 of present embodiment '-the phosphonuclease diagnostic reagent has:
Reagent I
Damping fluid 100mmol/l
Pyruvic acid 4mmol/l
Phosphoenolpyruvic acid 3mmol/l
Reduced coenzyme 0.25mmol/l
Pyruvic oxidase 12000U/l
Phosphoric acid enol pyruvic acid carboxylase 10000U/l
Malate dehydrogenase (malic acid dehydrogenase) 8000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Uridine list phosphoric acid 2mmol/l
Stablizer 10mmol/l
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, tested 5 '-volume ratio of phosphonuclease 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.
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+
Carbonic acid gas+hydrogen peroxide
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 5 '-the active size of phosphonuclease.
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. 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, phosphoenolpyruvic acid, reduced coenzyme, pyruvic oxidase, phosphoric acid enol pyruvic acid carboxylase, malate dehydrogenase (malic acid dehydrogenase), make it to take place the reaction of following principle:
Uridine list phosphoric acid+water
5 phosphonucleasesUridine+phosphate radical
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+
Carbonic acid gas+hydrogen peroxide
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 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 predominant wavelength 340nm, the speed that absorbancy descends, 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 claim 1 or 2 described mensuration 5 '-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
Uridine list phosphatase 11--10mmol/l
Pyruvic acid 1--10mmol/l
Phosphoenolpyruvic acid 1--20mmol/l
Reduced coenzyme 0.2--0.3mmol/l
Pyruvic oxidase 2000--20000U/l
Phosphoric acid enol pyruvic acid carboxylase 2000--20000U/l
Malate dehydrogenase (malic acid dehydrogenase) 2000--20000U/l
Stablizer 10--80% (cumulative volume)
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 can be 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: 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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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410064913 CN1760373A (en) | 2004-10-11 | 2004-10-11 | Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410064913 CN1760373A (en) | 2004-10-11 | 2004-10-11 | Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase |
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| CN1760373A true CN1760373A (en) | 2006-04-19 |
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| CN 200410064913 Pending CN1760373A (en) | 2004-10-11 | 2004-10-11 | Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase |
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