CN1769484A - 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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- CN1769484A CN1769484A CN 200410065607 CN200410065607A CN1769484A CN 1769484 A CN1769484 A CN 1769484A CN 200410065607 CN200410065607 CN 200410065607 CN 200410065607 A CN200410065607 A CN 200410065607A CN 1769484 A CN1769484 A CN 1769484A
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- adenosine deaminase
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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, adenosine triphosphate, phosphoenolpyruvate phosphatase, deacidized type coenzyme, ammonia kinase, pyruvate kinase, lactate 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, adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, ammonia kinase, pyruvate kinase, serum lactic dehydrogenase, make it to take place the reaction of following principle:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
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 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, the speed that predominant wavelength 340nm absorbancy descends draws 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 ammonia kinase, pyruvate kinase, lactic dehydrogenase enzyme reaction continuous monitoring method.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling ammonia kinase again, adenosine triphosphate is become adenosine diphosphate (ADP), adenosine diphosphate (ADP) and phosphoenolpyruvic acid produce pyruvic acid under the effect of pyruvate kinase, 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 the active size of adenosine deaminase.
The adenosine deaminase diagnosis reagent kit of realizing the inventive method can be single agent, comprising:
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Ammonia kinase 500--50000U/l
Pyruvate kinase 500--50000U/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 ammonia, adenosine diphosphate (ADP), pyruvic acid pollution more helped eliminating:
Reagent I
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Ammonia kinase 500--50000U/l
Pyruvate kinase 500--50000U/l
Serum lactic dehydrogenase 500--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, and adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, ammonia kinase, pyruvate kinase, serum lactic 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, adenosine diphosphate (ADP), pyruvic acid pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Stablizer 10--50mmol/l
Reagent II
Damping fluid 40--200mmol/l
Ammonia kinase 500--50000U/l
Pyruvate kinase 500--50000U/l
Serum lactic dehydrogenase 500--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Stablizer 10--50mmol/l
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, and adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme ammonia kinase, pyruvate kinase, serum lactic dehydrogenase etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, ammonia kinase, pyruvate kinase, serum lactic 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.
The pH scope of buffer reagent can be 6.0～11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphoric acid salt (Phosphate) damping fluid, trolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid, imidazoles (Imidazole) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.
Above reduced coenzyme can be reduced form nicotinamide coenzyme or derivatives thereofs such as NADPH, NADH or thio-NADH.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer 10～80% or 10～50mmol/l among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stablizer can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine or the salt etc.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the adenosine deaminase diagnosis reagent kit of the present invention of following system component relation is comparatively desirable:
Damping fluid 80--120mmol/l
Adenosine 2--10mmol/l
Adenosine triphosphate 2--8mmol/l
Phosphoenolpyruvic acid 2--8mmol/l
Reduced coenzyme 0.2--0.3mmo/l
Ammonia kinase 4000--10000U/l
Pyruvate kinase 1000--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 ammonia, adenosine diphosphate (ADP), pyruvic acid, the effect of eliminating inside and outside source ammonia, 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 ammonia, adenosine diphosphate (ADP), pyruvic acid, and all be the activity that results from adenosine deaminase at the needed ammonia of second half section time test activity of adenosine deaminase, adenosine diphosphate (ADP), pyruvic acid.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
The adenosine deaminase diagnosis reagent kit of present embodiment comprises:
Damping fluid 80mmol/l
Adenosine 2mmol/l
Adenosine triphosphate 2mmol/l
Phosphoenolpyruvic acid 2mmol/l
Reduced coenzyme 0.2mmo/l
Ammonia kinase 4000U/l
Pyruvate kinase 1000U/l
Serum lactic dehydrogenase 5000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, and the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
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 the active size of adenosine deaminase.
Present embodiment is used adenosine deaminase coupling ammonia kinase, pyruvate kinase, lactic dehydrogenase enzyme reaction continuous monitoring method.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling ammonia kinase again, adenosine triphosphate is become adenosine diphosphate (ADP), adenosine diphosphate (ADP) and phosphoenolpyruvic acid produce pyruvic acid under the effect of pyruvate kinase, 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 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
Adenosine triphosphate 5mmol/l
Phosphoenolpyruvic acid 5mmol/l
Reduced coenzyme 0.25mmo/l
Ammonia kinase 7000U/l
Pyruvate kinase 6000U/l
Serum lactic dehydrogenase 12000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Adenosine 6mmol/l
Stablizer 20mmol/l
When measuring activity of adenosine deaminase, temperature is controlled at 30 ℃, 15 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
Concrete determination step is:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
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 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
Adenosine triphosphate 8mmol/l
Phosphoenolpyruvic acid 8mmol/l
Reduced coenzyme 0.3mmo/l
Stablizer 20mmol/l
Reagent II
Damping fluid 120mmol/l
Ammonia kinase 10000U/l
Pyruvate kinase 10000U/l
Serum lactic dehydrogenase 20000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Adenosine 10mmol/l
Stablizer 20mmol/l
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, and the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
Concrete determination step is:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
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 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
Adenosine triphosphate 2mmol/l
Phosphoenolpyruvic acid 2mmol/l
Reduced coenzyme 0.3mmo/l
Stablizer 20mmol/l
Reagent II
Damping fluid 100mmol/l
Adenosine 4mmol/l
Ammonia kinase 10000U/l
Pyruvate kinase 8000U/l
Serum lactic dehydrogenase 18000U/l
Stablizer 50% (cumulative volume)
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, and the Direction of Reaction is negative reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-4180.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
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 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, adenosine triphosphate, phosphoenolpyruvic acid, reduced coenzyme, ammonia kinase, pyruvate kinase, serum lactic dehydrogenase, make it to take place the reaction of following principle:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+ammonia
Ammonia kinaseAdenosine diphosphate (ADP)+phosphamide
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 the active size of adenosine deaminase.
2. according to the method for the described mensuration activity of adenosine deaminase of claim 1, it is characterized in that: described step 2) be, the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detection is calculated the active size of adenosine deaminase in the speed that predominant wavelength 340nm absorbancy descends.
3, according to the method for claim 1 or 2 described mensuration activity of adenosine deaminase, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction times was controlled at 2-30 minute.
4, according to the method for claim 1 or 2 described mensuration activity of adenosine deaminase, it is characterized in that: the ratio control of sample and reagent is 1/10 to 1/250.
5. adenosine deaminase diagnosis reagent kit is grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Adenosine triphosphate 1--10mmol/l
Phosphoenolpyruvic acid 1--10mmol/l
Reduced coenzyme 0.15--0.3mmo/l
Ammonia kinase 500--50000U/l
Pyruvate kinase 500--50000U/l
Serum lactic dehydrogenase 500--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 200410065607 CN1769484A (en) | 2004-11-05 | 2004-11-05 | Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit |
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