CN101750383A - Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase - Google Patents
Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase Download PDFInfo
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- CN101750383A CN101750383A CN200810244278A CN200810244278A CN101750383A CN 101750383 A CN101750383 A CN 101750383A CN 200810244278 A CN200810244278 A CN 200810244278A CN 200810244278 A CN200810244278 A CN 200810244278A CN 101750383 A CN101750383 A CN 101750383A
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- adenosine deaminase
- stabilizing agent
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Abstract
The invention relates to a diagnostic reagent (kit) of adenosine deaminase, which utilizes enzyme colorimetry and an enzyme linkage method technology. Meanwhile, the invention also relates to a method for measuring the activity concentration of the adenosine deaminase and the formation and the components of the reagent, which belong to the technical fields of the inspection and the measurement of medicine. The reagent (kit) comprises the main components of a buffer solution, reduced coenzyme, adenosine, (indole-3-radical) pyruvic acid, calcium chloride, tryptophan dehydrogenase and a stabilizing agent; and a sample and the reagent generate a series of enzymatic reactions by mixing the sample and the reagent according to a certain volume ratio, then a reactant is put under an ultraviolet/visible light analyzer, and the descending speed of the absorbency of a position with the dominant wavelength of 340nm is detected, thereby measuring and calculating the magnitude of the activity concentration of the adenosine deaminase.
Description
Technical field
The present invention relates to a kind of adenosine deaminase diagnosing reagent (box), the invention still further relates to the method for measuring adenosine deaminase active density simultaneously, belong to medical test 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 deaminase inosine (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 enzymic colorimetric (Enzymatic ColorimetricMethod) and enzyme (even) united method (Couple Reaction) technology utilized, 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 the adenosine deaminase diagnosing reagent (box) in order to realize this method, adopt this reagent 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 of the present invention is as follows:
Adenosine+water gland guanosine deaminase inosine+ammonia
Ammonia+(indol-3-yl) pyruvic acid+reduced coenzyme tryptophane dehydrogenasa/Ca
2+
Tryptophane+coenzyme
This method is used adenosine deaminase (Adenosine Deaminase; EC 3.5.4.4) enzyme (idol) connection tryptophane dehydrogenasa (Tryptophan dehydrogenase; EC 1.4.1.19) enzymatic reaction continuous monitoring method.The reaction of adenosine deaminase enzymolysis adenosine produces ammonia, the effect of uniting the tryptophane dehydrogenasa again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, 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
Reduced coenzyme 0.25mmol/L
Tryptophane dehydrogenase 10 000U/L
Adenosine 6mmol/L
(indol-3-yl) pyruvic acid 9mmol/L
Lime chloride 9mmol/L
Adenosine deaminase diagnosing reagent of the present invention (box) can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, lime chloride.
Reagent (box) can be 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, reduced coenzyme, adenosine, (indol-3-yl) pyruvic acid, lime chloride.
Reagent 2
Damping fluid, stabilizing agent, tryptophane dehydrogenasa.
Reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, the position of lime chloride in reagent 1 or reagent 2 can not limit.Reagent (box) can be 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, reduced coenzyme, (indol-3-yl) pyruvic acid, lime chloride.
Reagent 2
Damping fluid, stabilizing agent, adenosine.
Reagent 3
Damping fluid, stabilizing agent, tryptophane dehydrogenasa.
Reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, the position of lime chloride in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be 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 reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.
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
Reduced coenzyme 0.25mmol/L
Tryptophane dehydrogenase 10 000U/L
Adenosine 6mmol/L
(indol-3-yl) pyruvic acid 9mmol/L
Lime chloride 9mmol/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 1.8 ± 0.7, 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 negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-4180.
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 descends, 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
Reduced coenzyme 0.25mmol/L
Adenosine 6mmol/L
(indol-3-yl) pyruvic acid 9mmol/L
Lime chloride 9mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Tryptophane dehydrogenase 10 000U/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 1.8 ± 0.7, 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 negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-2170.
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 descends, 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
Reduced coenzyme 0.25mmol/L
(indol-3-yl) pyruvic acid 9mmol/L
Lime chloride 9mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Adenosine 6mmol/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Tryptophane dehydrogenase 10 000U/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 1.8 ± 0.7, 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, and the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value-2170.
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 descends, 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 descend; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 700U/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 4%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.00046 ± 0.00023 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 enzymic colorimetric and enzyme-linked method technology, its method is as follows:
Adenosine+water gland guanosine deaminase inosine+ammonia
Ammonia+(indol-3-yl) pyruvic acid+reduced coenzyme tryptophane dehydrogenasa/Ca
2+
Tryptophane+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 descends, calculate the active concentration size result of adenosine deaminase.
2. an adenosine deaminase diagnosing reagent (box), principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Tryptophane dehydrogenase 10 00---80000U/L
Adenosine 1---50mmol/L
(indol-3-yl) pyruvic acid 1---50mmol/L
Lime chloride 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.Lime chloride can replace with other calcium salts.
It is characterized in that: reagent (box) can be 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 diagnosing reagent of claim 2 (box), it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, lime chloride.
4. according to the described adenosine deaminase diagnosing reagent of claim 2 (box), it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, lime chloride; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenosine, (indol-3-yl) pyruvic acid, lime chloride; Reagent 2 is made up of damping fluid, stabilizing agent, tryptophane dehydrogenasa.Reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, the position of lime chloride in reagent 1 or reagent 2 can not limit.
5. according to the described adenosine deaminase diagnosing reagent of claim 2 (box), it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, lime chloride; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenosine, (indol-3-yl) pyruvic acid, lime chloride; Reagent 2 is made up of damping fluid, stabilizing agent, E2, E3; Reagent 3 is made up of damping fluid, stabilizing agent, tryptophane dehydrogenasa.Reduced coenzyme, tryptophane dehydrogenasa, adenosine, (indol-3-yl) pyruvic acid, the position of lime chloride in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described adenosine deaminase diagnosing reagent of claim 2 (box), it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810244278A CN101750383A (en) | 2008-12-10 | 2008-12-10 | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810244278A CN101750383A (en) | 2008-12-10 | 2008-12-10 | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase |
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| CN101750383A true CN101750383A (en) | 2010-06-23 |
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| CN200810244278A Pending CN101750383A (en) | 2008-12-10 | 2008-12-10 | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase |
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Open date: 20100623 |