CN101169411A - Ammonia diagnosis/determination reagent kit and ammonia concentration determination method - Google Patents
Ammonia diagnosis/determination reagent kit and ammonia concentration determination method Download PDFInfo
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- CN101169411A CN101169411A CNA2006100972278A CN200610097227A CN101169411A CN 101169411 A CN101169411 A CN 101169411A CN A2006100972278 A CNA2006100972278 A CN A2006100972278A CN 200610097227 A CN200610097227 A CN 200610097227A CN 101169411 A CN101169411 A CN 101169411A
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- glyceraldehyde
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- phosphate
- stabilizing agent
- ammonia
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Abstract
The invention relates to an ammonia diagnosing/measuring reagent box which utilizes the technique of the enzyme contrasting color method and the enzyme jointing method, and belongs to the technical field of the medicine/food/environment test. The main components of the reagent box of the invention mainly include cushion liquid, coenzyme, glutamic acid, adentosine triphosphoric acid, glyceraldehyde-3-phosphate, glutamine synthetase, glyceraldehyde-3-phosphate dehydrogenase and stabilizing agent; the reagent box generates a series of enzymic reactions through the mixing of the samples and the reagent at a certain cubage rate, and then the reactants are positioned under an ultraviolet/visible light analyzer which tests the reducing speed of the absorbency at the main wave length of 340nm, thereby measuring the concentration size of the enzyme. The invention can absolutely get needed measuring result through the ultraviolet/visible light analyzer.
Description
Technical field
The present invention relates to a kind of ammonia diagnosis/determination kit, the invention still further relates to the method for measuring ammonia concentration simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
The ammonia method for measuring has microdiffusion, ion exchange process, enzyme process and ammonia electrode method etc.Use at present maximum methods and be enzyme process and based on the determination of blood ammonia instrument analytic approach of ion-selective electrode.
Diffusion method discharges NH after being the sample alkalization
3, the ammonia that discharges with acidometric titration, or form the two mercury amine of pale brown look iodate with the Nessler reaction and carry out colorimetric.These methods need alkalization, and endogenic ammonia forms and impacts, and its accuracy and precision are affected, and seldom use at present; Ion exchange process is more accurate than diffusion method, and CV is 8%～13%; Ion selective electrode method is to utilize NH
3Be diffused into electrode surface, the PH that causes electrode changes and measures, and the CV of this method is 3.5%～4.8%, recovery height.In conjunction with concrete actual, should be practical with the enzymatic assays.
The retrieval Chinese patent is only found 87105593.7 patented claims and is disclosed a kind of rapid freezing cup for blood ammonia determination, does not but find more satisfactory determination of blood ammonia method.
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 ammonia concentration, simultaneously, the present invention also will provide in order to realize the ammonia diagnosis/determination kit of this method, adopt this kit not only can be ultraviolet analyser or half, carry out ammonia concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Ammonia concentration determination method principle of the present invention is as follows:
Ammonium ion+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+adenosine diphosphate+phosphate radical
Phosphate radical+glyceraldehyde-3-phosphate+coenzyme
Glyceraldehyde-3-phosphate dehydrogenaseGlyceric acid-1,3-bis phosphoric acid+reduced coenzyme
This method is used glutamine synthelase (glutamine synthetase; EC 6.3.1.2) coupling glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase; EC 1.2.1.59) enzymatic reaction end-point method.Glutamine synthelase enzymolysis ammonia react produces phosphate radical, effect by the coupling glyceraldehyde-3-phosphate dehydrogenase again, 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 degree that reduced coenzyme rises in 340nm place absorbance, by measuring the degree that 340nm place absorbance rises, can calculate the concentration of ammonia.
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 ammonia diagnosis/determination kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glutamine synthelase 4000U/L
Glyceraldehyde-3-phosphate dehydrogenase 6000U/L
Glutamic acid 16mmol/L
Adenosine triphosphate 6mmol/L
Glyceraldehyde-3-phosphate 4mmol/L
Ammonia diagnosis/determination kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.
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, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate.
Reagent 2
Damping fluid, stabilizing agent, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.
Coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase 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, glutamic acid.
Reagent 2
Damping fluid, stabilizing agent, adenosine triphosphate, glyceraldehyde-3-phosphate.
Reagent 3
Damping fluid, stabilizing agent, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.
Coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase 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 ammonia concentration, 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 ammonia diagnosing/determining reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glutamine synthelase 4000U/L
Glyceraldehyde-3-phosphate dehydrogenase 6000U/L
Glutamic acid 16mmol/L
Adenosine triphosphate 6mmol/L
Glyceraldehyde-3-phosphate 4mmol/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 ammonia sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), about about 0 minute of time delay is about 5 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 degree that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of ammonia.
Embodiment two
The ammonia diagnosing/determining reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glutamic acid 12mmol/L
Adenosine triphosphate 8mmol/L
Glyceraldehyde-3-phosphate 6mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glutamine synthelase 6000U/L
Glyceraldehyde-3-phosphate 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 ammonia sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), about about 0 minute of time delay is about 5 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 degree that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of ammonia.
Embodiment three
The ammonia diagnosing/determining reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glutamic acid 20mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Adenosine triphosphate 4mmol/L
Glyceraldehyde-3-phosphate 8mmol/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glutamine synthelase 8000U/L
Glyceraldehyde-3-phosphate dehydrogenase 4000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring ammonia concentration, 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 ammonia 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 0 minute of time delay is about 5 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 degree that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of ammonia.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, and is easy to utilize.
Claims (6)
1. the method for measurement of concentration of the ammonia of an enzymic colorimetric, its method principle is as follows:
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbance rises, calculate the concentration measurement result of ammonia.
2. ammonia diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---50mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Glutamine synthelase 1000---80000U/L
Glyceraldehyde-3-phosphate dehydrogenase 1000---80000U/L
Glutamic acid 1---20mmol/L
Adenosine triphosphate 1---12mmol/L
Glyceraldehyde-3-phosphate 1---8mmol/L
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 ammonia diagnosis/determination kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.
4. according to the described ammonia diagnosis/determination kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate; Reagent 2 is made up of damping fluid, stabilizing agent, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.Coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described ammonia diagnosis/determination kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase; Reagent 1 is by damping fluid, stabilizing agent, coenzyme, glutamic acid, adenosine triphosphate, form; Reagent 2 is made up of damping fluid, stabilizing agent, glyceraldehyde-3-phosphate; Reagent 3 is made up of damping fluid, stabilizing agent, glutamine synthelase, glyceraldehyde-3-phosphate dehydrogenase.Coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate, glutamine synthelase, the position of glyceraldehyde-3-phosphate dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described ammonia diagnosis/determination 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 (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100972278A CN101169411A (en) | 2006-10-24 | 2006-10-24 | Ammonia diagnosis/determination reagent kit and ammonia concentration determination method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100972278A CN101169411A (en) | 2006-10-24 | 2006-10-24 | Ammonia diagnosis/determination reagent kit and ammonia concentration determination method |
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|---|---|
| CN101169411A true CN101169411A (en) | 2008-04-30 |
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|---|---|---|---|
| CNA2006100972278A Pending CN101169411A (en) | 2006-10-24 | 2006-10-24 | Ammonia diagnosis/determination reagent kit and ammonia concentration determination method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102087267A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Method and kit for measuring glycine |
| CN102087265A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Determination method of glycine and kit for determining glycine |
| CN102087289A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Method and kit for measuring glycine |
| CN102539704A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN102539702A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN102565332A (en) * | 2010-12-13 | 2012-07-11 | 苏州艾杰生物科技有限公司 | Determination method of ammonia (ammonia ions) and diagnosis/determination kit for ammonia (ammonia ions) |
| CN113075139A (en) * | 2021-03-29 | 2021-07-06 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
-
2006
- 2006-10-24 CN CNA2006100972278A patent/CN101169411A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102087267A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Method and kit for measuring glycine |
| CN102087265A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Determination method of glycine and kit for determining glycine |
| CN102087289A (en) * | 2009-12-04 | 2011-06-08 | 苏州艾杰生物科技有限公司 | Method and kit for measuring glycine |
| CN102539704A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN102539702A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN102565332A (en) * | 2010-12-13 | 2012-07-11 | 苏州艾杰生物科技有限公司 | Determination method of ammonia (ammonia ions) and diagnosis/determination kit for ammonia (ammonia ions) |
| CN113075139A (en) * | 2021-03-29 | 2021-07-06 | 迪瑞医疗科技股份有限公司 | Stable double-reagent blood ammonia determination kit |
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Open date: 20080430 |