CN101609009A - 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
- Publication number
- CN101609009A CN101609009A CNA2008101228221A CN200810122822A CN101609009A CN 101609009 A CN101609009 A CN 101609009A CN A2008101228221 A CNA2008101228221 A CN A2008101228221A CN 200810122822 A CN200810122822 A CN 200810122822A CN 101609009 A CN101609009 A CN 101609009A
- Authority
- CN
- China
- Prior art keywords
- reagent
- adenosine
- adenosine deaminase
- stabilizing agent
- coenzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of adenosine deaminase diagnosis reagent kit that utilizes 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, pyruvic acid, hydrogen peroxide, glycine oxidase, alanine 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 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 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 enzymic colorimetric (EnzymaticColorimetric Method) 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 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
Ammonia+pyruvic acid+hydrogen peroxide
Glycine oxidaseAlanine+water+oxygen
Alanine+water+coenzyme
Alanine dehydrogenaseAmmonia+pyruvic acid+reduced coenzyme
This method is used adenosine deaminase (Adenosine Deaminase; EC 3.5.4.4) enzyme (idol) connection glycine oxidase (glycine oxidase; EC X.X.X.X), alanine dehydrogenase (EC 1.4.3.19; EC1.4.1.1) enzymatic reaction continuous monitoring method.The reaction of adenosine deaminase enzymolysis adenosine produces ammonia, the effect of uniting glycine oxidase, alanine dehydrogenase again by (idol), coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged 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
Glycine oxidase 6000U/L
Alanine dehydrogenase 8000U/L
Adenosine 5mmol/L
Pyruvic acid 12mmol/L
Hydrogen peroxide 7mmol/L
Adenosine deaminase diagnosis reagent kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, hydrogen peroxide.
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, pyruvic acid, hydrogen peroxide.
Reagent 2
Damping fluid, stabilizing agent, glycine oxidase, alanine dehydrogenase.
Coenzyme, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, the position of hydrogen peroxide 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, pyruvic acid, hydrogen peroxide.
Reagent 2
Damping fluid, stabilizing agent, alanine dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, glycine oxidase.
Coenzyme, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, the position of hydrogen peroxide 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
Glycine oxidase 6000U/L
Alanine dehydrogenase 8000U/L
Adenosine 5mmol/L
Pyruvic acid 12mmol/L
Hydrogen peroxide 7mmol/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, 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 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 5mmol/L
Pyruvic acid 12mmol/L
Hydrogen peroxide 7mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glycine oxidase 6000U/L
Alanine 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, 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 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 5mmol/L
Pyruvic acid 12mmol/L
Hydrogen peroxide 7mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Alanine dehydrogenase 8000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glycine 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, and the Direction of Reaction is positive reaction (reaction of rising), 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 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)≤0.0005; 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 4%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); 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 principle is as follows:
Adenosine+water
Adenosine deaminaseInosine+ammonia
Ammonia+pyruvic acid+hydrogen peroxide
Glycine oxidaseAlanine+water+oxygen
Alanine+water+coenzyme
Alanine dehydrogenaseAmmonia+pyruvic 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
Glycine oxidase 1000---80000U/L
Alanine dehydrogenase 1000---80000U/L
Adenosine 1---50mmol/L
Pyruvic acid 1---50mmol/L
Hydrogen peroxide 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, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, hydrogen peroxide.
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, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, hydrogen peroxide; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, adenosine, pyruvic acid, hydrogen peroxide; Reagent 2 is made up of damping fluid, stabilizing agent, glycine oxidase, alanine dehydrogenase.Coenzyme, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, the position of hydrogen peroxide 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, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, hydrogen peroxide; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, adenosine, pyruvic acid, hydrogen peroxide; Reagent 2 is made up of damping fluid, stabilizing agent, alanine dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, glycine oxidase.Coenzyme, glycine oxidase, alanine dehydrogenase, adenosine, pyruvic acid, the position of hydrogen peroxide 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101228221A CN101609009A (en) | 2008-06-19 | 2008-06-19 | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101228221A CN101609009A (en) | 2008-06-19 | 2008-06-19 | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101609009A true CN101609009A (en) | 2009-12-23 |
Family
ID=41482838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008101228221A Pending CN101609009A (en) | 2008-06-19 | 2008-06-19 | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101609009A (en) |
-
2008
- 2008-06-19 CN CNA2008101228221A patent/CN101609009A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101750355A (en) | Adenosine deaminase diagnostic reagent (kit) and adenosine deaminase activity concentration measuring method | |
| CN101750354A (en) | Adenosine deaminase diagnostic reagent (kit) and adenosine deaminase activity concentration measuring method | |
| CN101096701A (en) | Adenosine deaminase diagnosing reagent case and method for detecting adenosine deaminase active density | |
| CN101620167A (en) | Adenosine deaminase diagnosis kit and method for determining adenosine deaminase activity concentration | |
| CN101609009A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method | |
| CN101750382A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101750381A (en) | Adenosine deaminase diagnostic reagent (kit) and adenosine deaminase activity concentration measuring method | |
| CN101750378A (en) | Adenosine deaminase diagnostic reagent (kit) and adenosine deaminase activity concentration measuring method | |
| CN101609010A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method | |
| CN101609011A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method | |
| CN101793740A (en) | Isocitric acid determination reagent (kit) and isocitric acid concentration determination method | |
| CN101762548A (en) | Adenosine deaminase diagnostic reagent (kit) and method for measuring activity concentration of adenosine deaminase | |
| CN101609012A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method | |
| CN101750379A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101750384A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101620168A (en) | Adenosine deaminase diagnosis kit and method for determining adenosine deaminase activity concentration | |
| CN101464263A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method | |
| CN101464264A (en) | Adenosine deaminase diagnosis reagent kit and adenosine deaminase active concentration determination method | |
| CN101750383A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101750380A (en) | Diagnostic reagent (kit) of adenosine deaminase and method for measuring activity concentration of adenosine deaminase | |
| CN101762547A (en) | Adenosine deaminase diagnostic reagent (kit) and method for measuring activity concentration of adenosine deaminase | |
| CN101620066A (en) | 5'nucleotidase diagnosis kit and method for determining 5'nucleotidase activity concentration | |
| CN101324565A (en) | Adenosine deaminase diagnosis reagent kit and method for determining adenosine deaminase activity concentration | |
| CN101793738A (en) | Isocitric acid determination reagent (kit) and isocitric acid concentration determination method | |
| CN101620069A (en) | Manganese ion diagnosis/determination kit and method for determining manganese ion concentration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| DD01 | Delivery of document by public notice |
Addressee: Aijie Biological Science & Technology Co., Ltd., Suzhou City Document name: Notification of before Expiration of Request of Examination as to Substance |
|
| DD01 | Delivery of document by public notice |
Addressee: Wang Erzhong Document name: Notification that Application Deemed to be Withdrawn |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20091223 |