CN1778964A - Determination of 5'-nucleotidase activity and its diagnostic reagent kit of 5'-nucleotidase - Google Patents
Determination of 5'-nucleotidase activity and its diagnostic reagent kit of 5'-nucleotidase Download PDFInfo
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- CN1778964A CN1778964A CN 200410065565 CN200410065565A CN1778964A CN 1778964 A CN1778964 A CN 1778964A CN 200410065565 CN200410065565 CN 200410065565 CN 200410065565 A CN200410065565 A CN 200410065565A CN 1778964 A CN1778964 A CN 1778964A
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Abstract
The invention is about a method of measuring the activation of 5'-phosphonuclease, and it also concerns the reagent box of 5'-phosphonucleasediagnosis. This invention belongs to the field of medical testing and measuring technology. The reagent box is consisted of buffer solution, carnine monophosphate, adenosine triphosphate, phosphoenolpyruvate, reduced coenzyme, carnine kinase, pyruvate kinase, lactate dehydrogenase and stabilizer. Firstly, we cause an enzyme-coupled reaction through mixing the sample and the reagent according to a certain proportion of volume; secondly, put the final reactant under the biochemical analyzer and test the absorbance variational situation (speed) of dominant wavelength; then we can get theactivation of 5'-phosphonuclease. By using this invention, we can get the necessary measuring result with high sensitiveness and fine precision through biochemical analyzer, and the result would not be contaminated by material of internal and exogenous sources. Thus, this method can be conveniently promoted and applied.
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 adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, Inosine kinase, pyruvate kinase, serum lactic dehydrogenase, make it to take place the reaction of following principle:
Inosine list phosphoric acid+water
5 '-phosphonucleaseInosine+single phosphoric acid
Adenosine triphosphate+inosine
Inosine kinaseAdenosine diphosphate (ADP)+inosine list phosphoric acid
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate
+ pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic 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.
This method uses 5 '-phosphonuclease coupling Inosine kinase (EC 2.7.1.73), pyruvate kinase, lactic dehydrogenase enzyme reaction continuous monitoring method.5 '-phosphonuclease hydrolysis inosine list phosphoric acid generation inosine, Inosine kinase catalysis inosine and adenosine triphosphate produce adenosine diphosphate (ADP) then, pyruvate kinase is catalysis adenosine diphosphate (ADP) and phosphoenolpyruvic acid generation pyruvic acid again, pyruvic acid is the effect by the coupling serum lactic 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 5 '-phosphonuclease active big or small.
5 of realization the inventive method '-the phosphonuclease diagnostic kit can be single agent, comprising:
Damping fluid 40---200mmol/l
Inosine list phosphoric acid 0.5---50mmol/l
Adenosine triphosphate 0.5---50mmol/l
Phosphoenolpyruvic acid 0.5---50mmol/l
Reduced coenzyme 0.15---0.3mmol/l
Inosine kinase 500---50000U/l
Pyruvate kinase 500---20000U/l
Serum lactic 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 diphosphate (ADP), pyruvic acid pollution more helped eliminating:
Reagent I
Damping fluid 40---200mmol/l
Adenosine triphosphate 0.5---50mmol/l
Phosphoenolpyruvic acid 0.5---50mmol/l
Reduced coenzyme 0.15---0.3mmol/l
Inosine kinase 500---50000U/l
Pyruvate kinase 500---20000U/l
Serum lactic dehydrogenase 500---50000U/l
Stablizer 10---80% (cumulative volume)
Reagent II
Damping fluid 40---200mmol/l
Inosine list phosphoric acid 0.5---50mmol/l
Stablizer 0---50mmol/l
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, Inosine kinase, pyruvate kinase, serum lactic dehydrogenase etc. can be placed on reagent II, reagent II composition wherein, inosine list phosphoric acid also can be placed on reagent I, so can form multiple formulations, not describe in detail one by one at this.
Reagent can also be made into following three reagent, not only more help eliminating inside and outside source adenosine diphosphate (ADP), pyruvic acid pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40---200mmol/l
Adenosine triphosphate 0.5---50mmol/l
Phosphoenolpyruvic acid 0.5---50mmol/l
Reduced coenzyme 0.15---0.3mmol/l
Stablizer 10---50mmol/l
Reagent II
Damping fluid 40---200mmol/l
Inosine kinase 500---50000U/l
Pyruvate kinase 500---20000U/l
Serum lactic dehydrogenase 500---50000U/l
Stablizer 10---80% (cumulative volume)
Reagent III
Damping fluid 40---200mmol/l
Inosine list phosphoric acid 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 adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, Inosine kinase, pyruvate kinase, serum lactic dehydrogenase etc. also can be placed among reagent I or the reagent III.Reagent III composition wherein, inosine list phosphoric acid also can be placed among reagent I or the reagent II, so can form multiple formulations, does not describe in detail one by one.
The pH scope of buffer reagent can be 6.0～11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, 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., (for example can also be imidazoles/hydrochloride buffer 6.2-7.8 but be not limited only to these damping fluids; Sodium phosphate dibasic citrate buffer solution 5.0-8.0; Citric acid sodium citrate buffer solution 5.0-6.6; Phosphate buffered saline buffer 6.0-8.0; Sodium phosphate dibasic-potassium phosphate buffer 6.0-8.0; Potassium primary phosphate sodium hydrate buffer solution 6.0-8.0; Veronal sodium-hydrochloride buffer 6.8-9.6; Tris hydrochloride buffer 7.0-9.0; Boric acid borate buffer solution 7.4-9.0; Glycine-sodium hydrate buffer solution 8.6-10.6; Sand-sodium hydrate buffer solution 9.2-10.0; Yellow soda ash-sodium bicarbonate buffer liquid 8.8-10.6 (Ca
2+, Mg
2+Must not use when existing); PBS damping fluid 7.0-7.6 etc.).
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 that keeps reagent, so that standing storage, above single agent, the reagent I of two agent, the reagent I of reagent II or three doses, reagent II, usually add stablizer 10～80% or 10～50mmol/l among the reagent III, stablizer can be an ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, in sulfuric acid amine or the salt etc. one or more can also be at thioglycol, adenosine diphosphate (ADP), bovine serum albumin, carbonate, cholate, dextran, ethylenediamine tetraacetic acid (EDTA), flavin adenine dinucleotide, vitamin B2 phosphate, glucose, glutaminate, lactose, N.F,USP MANNITOL, sucrose, sodium-chlor, select in the succinate etc.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, the present invention 5 of following system component relation '-the phosphonuclease diagnostic kit is comparatively desirable:
Damping fluid 80---120mmol/l
Inosine list phosphatase 11---10mmol/l
Adenosine triphosphate 2---8mmol/l
Phosphoenolpyruvic acid 2---8mmol/l
Reduced coenzyme 0.2---0.3mmol/l
Inosine kinase 4000---10000U/l
Pyruvate kinase 6000---10000U/l
Serum lactic dehydrogenase 5000---20000U/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 adenosine diphosphate (ADP), pyruvic acid, the effect of eliminating inside and outside source adenosine diphosphate (ADP), pyruvic acid occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source adenosine diphosphate (ADP), pyruvic acid, and second half section time test 5 '-the needed adenosine diphosphate (ADP) of activity of 5 '-nucleotidase, pyruvic acid 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
Inosine list phosphatase 11 mmol/l
Adenosine triphosphate 2mmol/l
Phosphoenolpyruvic acid 2mmol/l
Reduced coenzyme 0.2mmol/l
Inosine kinase 4000U/l
Pyruvate kinase 6000U/l
Serum lactic 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 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:
Inosine list phosphoric acid+water
5 '-phosphonucleaseInosine+single phosphoric acid
Adenosine triphosphate+inosine
Inosine kinaseAdenosine diphosphate (ADP)+inosine list phosphoric acid
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate
+ pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic 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 Inosine kinase (EC 2.7.1.73), pyruvate kinase, lactic dehydrogenase enzyme reaction continuous monitoring method.5 '-phosphonuclease hydrolysis inosine list phosphoric acid generation inosine, Inosine kinase catalysis inosine and adenosine triphosphate produce adenosine diphosphate (ADP) then, pyruvate kinase is catalysis adenosine diphosphate (ADP) and phosphoenolpyruvic acid generation pyruvic acid again, pyruvic acid is the effect by the coupling serum lactic 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 5 '-phosphonuclease active big or small.
Embodiment two (two agent)
5 of present embodiment '-the phosphonuclease diagnostic reagent has:
Damping fluid 100mmol/l
Adenosine triphosphate 5mmol/l
Phosphoenolpyruvic acid 5mmol/l
Reduced coenzyme 0.25mmol/l
Inosine kinase 7000U/l
Pyruvate kinase 8000U/l
Serum lactic dehydrogenase 13000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Inosine list phosphoric acid 6mmol/l
Stablizer 20mmol/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:
Inosine list phosphoric acid+water
5 '-phosphonucleaseInosine+single phosphoric acid
Adenosine triphosphate+inosine
Inosine kinaseAdenosine diphosphate (ADP)+inosine list phosphoric acid
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate
+ pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic 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
Adenosine triphosphate 8mmol/l
Phosphoenolpyruvic acid 8mmol/l
Reduced coenzyme 0.3mmol/l
Stablizer 20mmol/l
Reagent II
Damping fluid 120mmol/l
Inosine kinase 10000U/l
Pyruvate kinase 10000U/l
Serum lactic dehydrogenase 20000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Inosine list phosphatase 11 0mmol/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, 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:
Inosine list phosphoric acid+water
5 '-phosphonucleaseInosine+single phosphoric acid
Adenosine triphosphate+inosine
Inosine kinaseAdenosine diphosphate (ADP)+inosine list phosphoric acid
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate
+ pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic 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
Phosphoenolpyruvic acid 2mmol/l
Reduced coenzyme 0.3mmol/l
Inosine kinase 8000U/l
Pyruvate kinase 10000U/l
Serum lactic dehydrogenase 20000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Inosine list phosphoric acid 3mmol/l
Adenosine triphosphate 4mmol/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, 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:
Inosine list phosphoric acid+water
5 '-phosphonucleaseInosine+single phosphoric acid
Adenosine triphosphate+inosine
Inosine kinaseAdenosine diphosphate (ADP)+inosine list phosphoric acid
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate
+ pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic 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 adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, Inosine kinase, pyruvate kinase, serum lactic dehydrogenase, make it to take place the reaction of following principle:
Inosine list phosphoric acid+water
5 '-phosphonucleaseInosine+single phosphoric acid
Adenosine triphosphate+inosine
Inosine kinaseAdenosine diphosphate (ADP)+inosine list phosphoric acid
Adenosine diphosphate (ADP)+phosphoenolpyruvic acid
Pyruvate kinaseAdenosine triphosphate
+ pyruvic acid
Pyruvic acid+reduced coenzyme
Serum lactic dehydrogenaseLactic 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 the speed that predominant wavelength 340nm 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 1/250.
5. one kind 5 '-the phosphonuclease diagnostic kit, be grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Inosine list phosphoric acid 0.5--50mmol/l
Adenosine triphosphate 0.5--50mmol/l
Phosphoenolpyruvic acid 0.5--50mmol/l
Reduced coenzyme 0.15--0.3mmol/l
Inosine kinase 500--50000U/l
Pyruvate kinase 500--20000U/l
Serum lactic 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 or the salt.
According to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: the pH scope of described buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.
9, according to claim 5 or 6 described 5 '-the phosphonuclease diagnostic kit, it is characterized in that: 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108548782A (en) * | 2018-04-18 | 2018-09-18 | 易尚明天科技有限公司 | The active detection method of 5'-NT and kinetics quantitative detection instrument |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108548782A (en) * | 2018-04-18 | 2018-09-18 | 易尚明天科技有限公司 | The active detection method of 5'-NT and kinetics quantitative detection instrument |
| CN108548782B (en) * | 2018-04-18 | 2020-12-04 | 易尚明天科技有限公司 | Detection method of 5' -nucleotidase activity and reaction kinetics quantitative detector |
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