CN101609011A - Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method - Google Patents
Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method Download PDFInfo
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- CN101609011A CN101609011A CNA2008101228240A CN200810122824A CN101609011A CN 101609011 A CN101609011 A CN 101609011A CN A2008101228240 A CNA2008101228240 A CN A2008101228240A CN 200810122824 A CN200810122824 A CN 200810122824A CN 101609011 A CN101609011 A CN 101609011A
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- adenosine deaminase
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Abstract
The present invention relates to a kind of adenosine deaminase diagnosis reagent kit that utilizes enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method technology, the invention still further relates to the method principle of measuring adenosine deaminase active density, the composition and the composition of reagent simultaneously, belong to medical test determination techniques field.Kit principal ingredient of the present invention comprises: damping fluid, coenzyme, adenosine, hydrogen peroxide, 2-oxoglutaric acid, dglutamic oxidase, glutamte dehydrogenase and stabilizing agent; By sample is mixed by certain volume ratio with reagent, make it to take place series of enzymatic reactions, again reactant is placed under the ultraviolet analyser, detect the speed that predominant wavelength 340nm place absorbance rises, thereby calculate the active concentration size of adenosine deaminase.
Description
Technical field
The present invention relates to a kind of adenosine deaminase diagnosis reagent kit, the invention still further relates to the method for measuring adenosine deaminase active density simultaneously, belong to check determination techniques field.
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 antidiastole, severe combined immunodeficiency disease (SCID), acute and chronic hepatitis, 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 ultraviolet light method at present in the world, its measuring principle is: adenosine (white crystalline powder)+water (H
2O)
Adenosine deaminaseInosine (Inosine)+ammonium ion (NH
4 +), 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 absorbance of mensuration.Yet the 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.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzyme cycle amplification method (EnzymaticRecycling Method) of utilizing, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for adenosine deaminase active density, simultaneously, the present invention also will provide in order to realize the adenosine deaminase diagnosis reagent kit of this method, adopt this kit not only can be ultraviolet analyser or half, carry out detecting adenosine deaminase active density on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Adenosine deaminase active concentration determination method principle of the present invention is as follows:
Adenosine+water
Adenosine deaminaseInosine+ammonia
Hydrogen peroxide+ammonia+2-oxoglutaric acid
Dglutamic oxidaseGlutamic acid+oxygen+water
Glutamic acid+water+coenzyme
Glutamte dehydrogenaseAmmonia+2-oxoglutaric acid+
Reduced coenzyme
This method is used adenosine deaminase (Adenosine Deaminase; EC 3.5.4.4) enzyme (idol) connection dglutamic oxidase (Glutamate oxidase; EC 1.4.3.7EC 1.4.3.11), glutamte dehydrogenase (Glutamate Dehydrogenase; EC 1.4.1.2, EC 1.4.1.3, EC 1.4.1.4) the enzymatic reaction continuous monitoring method.Adenosine deaminase is as the effect enzyme, and function is that the adenosine enzymolysis is produced ammonia.Dglutamic oxidase can become glutamic acid with the ammonia effect; Glutamte dehydrogenase is as cyclophorase: its enzymatic catalysis makes glutamic acid produce ammonia, and it is recycling that ammonia constantly is repeated, and constantly produces glutamic acid simultaneously.Glutamte dehydrogenase is again as the colour developing enzyme, under the glutamic acid substrate-function, with coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place at the 340nm place, thereby measured the speed that reduced coenzyme rises in 340nm place absorbance, by measuring the speed that 340nm place absorbance rises, can calculate the active concentration size of adenosine deaminase.
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 diagnosing reagent of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Dglutamic oxidase 6000U/L
Glutamte dehydrogenase 8000U/L
Adenosine 6mmol/L
Hydrogen peroxide 5mmol/L
2-oxoglutaric acid 5mmol/L
Adenosine deaminase diagnosis reagent kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, 2-oxoglutaric acid.
Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme, adenosine, hydrogen peroxide, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, dglutamic oxidase, glutamte dehydrogenase.
Coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1 or reagent 2 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme, adenosine, hydrogen peroxide, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, glutamte dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, dglutamic oxidase.
Coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
No matter be single agent, two agent or three doses, the present invention measures the method for adenosine deaminase active density, and its coenzyme can be NADP
+, NAD
+Or thio-NAD
+In a kind of.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The adenosine deaminase diagnosing reagent of present embodiment is a single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Dglutamic oxidase 6000U/L
Glutamte dehydrogenase 8000U/L
Adenosine 6mmol/L
Hydrogen peroxide 5mmol/L
2-oxoglutaric acid 5mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance rises, thereby calculates the active concentration size of adenosine deaminase.
Embodiment two
The adenosine deaminase diagnosing reagent of present embodiment is a double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Adenosine 6mmol/L
Hydrogen peroxide 5mmol/L
2-oxoglutaric acid 5mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Dglutamic oxidase 6000U/L
Glutamte dehydrogenase 8000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance rises, thereby calculates the active concentration size of adenosine deaminase.
Embodiment three
The adenosine deaminase diagnosing reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Adenosine 6mmol/L
Hydrogen peroxide 5mmol/L
2-oxoglutaric acid 5mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutamte dehydrogenase 8000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Dglutamic oxidase 6000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring adenosine deaminase active density, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested adenosine deaminase sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is positive reaction (reaction of rising), and about about 1 minute of time delay is about 2 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 340nm absorbance rises, thereby calculates the active concentration size of adenosine deaminase.
The applicant adopts other assay methods of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.0006; Absorbance time response curve should linearly rise; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 500U/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.005 ± 0.002 Δ A/min/U/L; Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, the linear range broadness, and stationary phase is long, is enough to easy to utilize.
Claims (6)
1. adenosine deaminase active concentration determination method that utilizes enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Adenosine+water
Adenosine deaminaseInosine+ammonia
Hydrogen peroxide+ammonia+2-oxoglutaric acid
Dglutamic oxidaseGlutamic acid+oxygen+water
Glutamic acid+water+coenzyme
Glutamte dehydrogenaseAmmonia+2-oxoglutaric acid+
Reduced coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance rises, calculate the active concentration size measurement result of adenosine deaminase.
2. adenosine deaminase diagnosis reagent kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Dglutamic oxidase 1000---80000U/L
Glutamte dehydrogenase 1000---80000U/L
Adenosine 1---50mmol/L
Hydrogen peroxide 1---50mmol/L
2-oxoglutaric acid 1---50mmol/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.
It is characterized in that: kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
3. according to the described adenosine deaminase diagnosis reagent kit of claim 2, it is characterized in that: form single agent reagent by damping fluid, stabilizing agent, coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, 2-oxoglutaric acid.
4. according to the described adenosine deaminase diagnosis reagent kit of claim 2, it is characterized in that: form two agent reagent by damping fluid, stabilizing agent, coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, adenosine, hydrogen peroxide, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, dglutamic oxidase, glutamte dehydrogenase.Coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1 or reagent 2 can not limit.
5. according to the described adenosine deaminase diagnosis reagent kit of claim 2, it is characterized in that: form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, adenosine, hydrogen peroxide, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, glutamte dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, dglutamic oxidase.Coenzyme, dglutamic oxidase, glutamte dehydrogenase, adenosine, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described adenosine deaminase diagnosis reagent kit of claim 2, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethyleneglycol) and at least one of the preservatives.
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|---|---|---|---|
| CNA2008101228240A CN101609011A (en) | 2008-06-19 | 2008-06-19 | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method |
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|---|---|---|---|
| CNA2008101228240A CN101609011A (en) | 2008-06-19 | 2008-06-19 | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method |
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| Publication Number | Publication Date |
|---|---|
| CN101609011A true CN101609011A (en) | 2009-12-23 |
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ID=41482840
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|---|---|---|---|
| CNA2008101228240A Pending CN101609011A (en) | 2008-06-19 | 2008-06-19 | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method |
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|---|---|
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Open date: 20091223 |