CN1749411A - Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase - Google Patents
Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase Download PDFInfo
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- CN1749411A CN1749411A CN 200410041966 CN200410041966A CN1749411A CN 1749411 A CN1749411 A CN 1749411A CN 200410041966 CN200410041966 CN 200410041966 CN 200410041966 A CN200410041966 A CN 200410041966A CN 1749411 A CN1749411 A CN 1749411A
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- phosphonuclease
- adenosine
- nucleotidase
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Abstract
The present invention relates to 5'-nucleotidase activity detecting method and 5'-nucleotidase diagnosing reagent kit in medical detecting technology. The 5'-nucleotidase diagnosing reagent kit includes buffering liquid, adenosine monophosphate, monovalent phosphate, adenylate deaminase, nucleotide phosphorylase, xanthine oxidase, alcohol, hydrogen peroxidase, oxidation type coenzyme, aldehyde dehydrogenase and stabilizer. Through mixing the sample and reagent in certain volume ratio to produce enzyme coupling reaction, and detecting in biochemical analyzer the main wavelength absorbency change speed, the activity of 5'-nucleotidase is calculated. The present invention can obtain the measurement result in biochemical analyzer in high sensitivity, high precision and no contamination of various foreign and internal matters.
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
The technical problem to be solved in the present invention is: 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 adenosine monophosphate, adenosine deaminase, monophosphate, Phosphatase, nucleotide, XOD, ethanol, catalase, oxidized coenzyme, aldehyde dehydrogenase, make it to take place the reaction of following principle:
Adenosine monophosphate+water
5 '-phosphonucleaseAdenosine+single phosphoric acid
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Inosine+monophosphate
Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen
XODUrate+hydrogen peroxide
Hydrogen peroxide+ethanol
CatalaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
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.
This method uses 5 '-enzyme linked reaction systems such as phosphonuclease, adenosine deaminase, Phosphatase, nucleotide, XOD, catalase, aldehyde dehydrogenase, oxidized coenzyme is reduced into reducibility coenzyme the most at last, because reducibility coenzyme has absorption peak at wavelength 340nm, the increase degree of therefore testing wavelength 340nm absorption peak can directly reflect 5 '-size of activity of 5 '-nucleotidase.The advantage of this enzyme linked reaction system has:
Its utilize to measure growing amount that oxidized coenzyme is reduced into reducibility coenzyme and reflects 5 '-activity of 5 '-nucleotidase, the stability of oxidized coenzyme in solution is higher much than reducibility coenzyme, so this system is stable fine.
The reacted constituent that this enzyme linked reaction system forms all adds, and is not subjected to the pollution of inside and outside source material, and test result is accurate.
Be used to realize 5 of the inventive method '-the phosphonuclease diagnostic kit can be single agent, comprising:
Damping fluid 40--200mmol/l
Adenosine monophosphate 1--50mmol/l
Adenosine deaminase 500--50000U/l
Monophosphate 0.2--10mmol/l
Phosphatase, nucleotide 500--50000U/l
XOD 500--50000U/l
Ethanol 1--30mmol/l
Catalase 500--50000U/l
Oxidized coenzyme 0.5--20mmol/l
Aldehyde dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Also above single agent following pair of agent be can be made into, inside and outside source adenosine, inosine, hypoxanthic pollution more helped eliminating:
Reagent I
Damping fluid 40--200mmol/l
Adenosine deaminase 500--50000U/l
Monophosphate 0.2--10mmol/l
Phosphatase, nucleotide 500--50000U/l
XOD 500--50000U/l
Ethanol 1--30mmol/l
Catalase 500--50000U/l
Oxidized coenzyme 0.5--20mmol/l
Aldehyde dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Adenosine monophosphate 1--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 ethanol, catalase, aldehyde dehydrogenase etc. can be placed on reagent II, so can form multiple formulations, do not describe in detail one by one.
Reagent can also be made into following three reagent, not only more help eliminating inside and outside source adenosine, inosine, hypoxanthic pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Monophosphate 0.2--10mmol/l
Ethanol 1--30mmol/l
Oxidized coenzyme 0.5--20mmol/l
Stablizer 10--50mmol/l
Reagent II
Damping fluid 40--200mmol/l
Adenosine deaminase 500--50000U/l
Phosphatase, nucleotide 500--50000U/l
XOD 500--50000U/l
Catalase 500--50000U/l
Aldehyde dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Adenosine monophosphate 1--50mmol/l
Stablizer 10--50mmol/l
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, monophosphate, ethanol, oxidized coenzyme etc. can be placed among the reagent II, reagent II composition wherein, adenosine deaminase, Phosphatase, nucleotide, XOD, catalase, aldehyde dehydrogenase etc. also can be placed among the reagent I, so can form multiple formulations, not describe in detail one by one.
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 or glycylglycine (Glycylglycine) damping fluid etc.
Above oxidized coenzyme can be NADP
+, NAD
+Or thio-NAD
+Deng reduced form nicotinamide coenzyme or derivatives thereof.
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, salt or the adenosine diphosphate (ADP) 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
Adenosine monophosphate 1--5mmol/l
Adenosine deaminase 5000--10000U/l
Monophosphate 2--10mmol/l
Phosphatase, nucleotide 5000--10000U/l
XOD 5000--10000U/l
Ethanol 3--12mmol/l
Catalase 5000--10000U/l
Oxidized coenzyme 1--5mmol/l
Aldehyde dehydrogenase 5000--10000U/l
Stablizer 10--80% (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 to eliminate inside and outside source adenosine, inosine, hypoxanthic pollution, eliminate the first half that inside and outside source adenosine, inosine, hypoxanthic effect occur in the entire reaction time period, be consumed totally at time second half section contaminated inside and outside source adenosine, inosine, xanthoglobulin, and second half section time test 5 '-the needed acetaldehyde of activity of 5 '-nucleotidase all be result from 5 '-activity of phosphonuclease.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
5 of present embodiment '-the phosphonuclease diagnostic kit comprises:
Damping fluid 80mmol/l
Adenosine monophosphate 1mmol/l
Adenosine deaminase 5000U/l
Monophosphate 2mmol/l
Phosphatase, nucleotide 5000U/l
XOD 5000U/l
Ethanol 3mmol/l
Catalase 5000U/l
Oxidized coenzyme 1mmol/l
Aldehyde dehydrogenase 5000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ 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 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:
Adenosine monophosphate+water
5 '-phosphonucleaseAdenosine+single phosphoric acid
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Inosine+monophosphate
Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen
XODUrate+hydrogen peroxide
Hydrogen peroxide+ethanol
CatalaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.
This method uses 5 '-enzyme linked reaction systems such as phosphonuclease, adenosine deaminase, Phosphatase, nucleotide, XOD, catalase, aldehyde dehydrogenase, oxidized coenzyme is reduced into reducibility coenzyme the most at last, because reducibility coenzyme has absorption peak at wavelength 340nm, the increase degree of therefore testing wavelength 340nm absorption peak can directly reflect 5 '-size of activity of 5 '-nucleotidase.
Embodiment two (two agent)
5 of present embodiment '-the phosphonuclease diagnostic reagent has:
Reagent I
Damping fluid 100mmol/l
Adenosine deaminase 8000U/l
Monophosphate 6mmol/l
Phosphatase, nucleotide 8000U/l
XOD 8000U/l
Ethanol 8mmol/l
Catalase 8000U/l
Oxidized coenzyme 3mmol/l
Aldehyde dehydrogenase 8000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine monophosphate 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 positive reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
Concrete determination step is:
Adenosine monophosphate+water
5 '-phosphonucleaseAdenosine+single phosphoric acid
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Inosine+monophosphate
Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen
XODUrate+hydrogen peroxide
Hydrogen peroxide+ethanol
CatalaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.
The reaction times of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
5 of present embodiment '-the phosphonuclease diagnostic reagent is three doses, has:
Reagent I
Damping fluid 120mmol/l
Monophosphate 10mmol/l
Ethanol 12mmol/l
Oxidized coenzyme 5mmol/l
Stablizer 50mmol/l
Reagent II
Damping fluid 120mmol/l
Adenosine deaminase 10000U/l
Phosphatase, nucleotide 10000U/l
XOD 10000U/l
Catalase 10000U/l
Aldehyde dehydrogenase 10000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Adenosine monophosphate 5mmol/l
Stablizer 50mmol/l
On automatic clinical chemistry analyzer, set: 25 ℃ 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.
Concrete determination step is:
Adenosine monophosphate+water
5 '-phosphonucleaseAdenosine+single phosphoric acid
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Inosine+monophosphate
Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen
XODUrate+hydrogen peroxide
Hydrogen peroxide+ethanol
CatalaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy rises, thereby calculate 5 '-the active size of phosphonuclease.
The reaction times of each reactions steps was controlled at 10 minutes.
Embodiment four
5 of present embodiment '-the phosphonuclease diagnostic reagent has:
Reagent I
Damping fluid 100mmol/l
Adenosine deaminase 12000U/l
Monophosphate 5mmol/l
Phosphatase, nucleotide 12000U/l
Xanthine oxidation 12000U/l
Ethanol 8mmol/l
Hydrogen peroxidase 12 000U/l
Oxidized coenzyme 3mmol/l
Aldehyde dehydrogenase 12000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine monophosphate 3mmol/l
Stablizer 40mmol/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 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:
Adenosine monophosphate+water
5 '-phosphonucleaseAdenosine+single phosphoric acid
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Inosine+monophosphate
Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen
XODUrate+hydrogen peroxide
Hydrogen peroxide+ethanol
CatalaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy rises, 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 adenosine monophosphate, adenosine deaminase, monophosphate, Phosphatase, nucleotide, XOD, ethanol, catalase, oxidized coenzyme, aldehyde dehydrogenase, make it to take place following reaction:
Adenosine monophosphate+water
5 '-phosphonucleaseAdenosine+single phosphoric acid
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Inosine+monophosphate
Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen
XODUrate+hydrogen peroxide
Hydrogen peroxide+ethanol
CatalaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
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) for the end reaction thing being placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect predominant wavelength 340nm, more than the test commplementary wave length 405nm, the speed that absorbancy rises, calculate 5 '-the active size of phosphonuclease.
3, according to claim 1 or 2 described 5 '-the activity of 5 '-nucleotidase measuring method, 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 3 described 5 '-the activity of 5 '-nucleotidase measuring method, it is characterized in that: tested 5 '-ratio control of phosphonuclease sample and reagent is 1/10 to 1/250.
5. one kind 5 '-the phosphonuclease diagnostic kit, be grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Adenosine monophosphate 1--50mmol/l
Adenosine deaminase 500--50000U/l
Monophosphate 0.2--10mmol/l
Phosphatase, nucleotide 500--50000U/l
XOD 500--50000U/l
Ethanol 1--30mmol/l
Catalase 500--50000U/l
Oxidized coenzyme 0.5--20mmol/l
Aldehyde dehydrogenase 500--50000U/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, salt or the adenosine diphosphate (ADP).
According to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: 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 or the glycylglycine damping fluid.
9, according to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: described oxidized coenzyme is NADP
+, NAD
+, thio-NAD
+A kind of in the reduced form nicotinamide coenzyme or derivatives thereof.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410041966 CN1749411A (en) | 2004-09-14 | 2004-09-14 | Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410041966 CN1749411A (en) | 2004-09-14 | 2004-09-14 | Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase |
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|---|---|
| CN1749411A true CN1749411A (en) | 2006-03-22 |
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324630A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit |
| CN109112181A (en) * | 2017-06-24 | 2019-01-01 | 浙江亚培生物技术有限公司 | A kind of 5`- activity of 5 '-nucleotidase measuring method and 5`- nucleotidase detection kit |
-
2004
- 2004-09-14 CN CN 200410041966 patent/CN1749411A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324630A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit |
| CN109112181A (en) * | 2017-06-24 | 2019-01-01 | 浙江亚培生物技术有限公司 | A kind of 5`- activity of 5 '-nucleotidase measuring method and 5`- nucleotidase detection kit |
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