CN101464341A - Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method - Google Patents
Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method Download PDFInfo
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- CN101464341A CN101464341A CNA2007101921348A CN200710192134A CN101464341A CN 101464341 A CN101464341 A CN 101464341A CN A2007101921348 A CNA2007101921348 A CN A2007101921348A CN 200710192134 A CN200710192134 A CN 200710192134A CN 101464341 A CN101464341 A CN 101464341A
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- glyceraldehyde
- phosphate
- ammonia
- coenzyme
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Abstract
The invention relates to a kit for diagnosing/measuring ammonia (ions) by utilizing the technologies of the enzymic colorimetry and the enzyme linked immunosorbent assay. The invention further relates to a method, a principle and the composition and the components of a reagent for measuring the concentration of ammonia (ions), and belongs to the technical field of medical/food/environmental inspection and measurement. The main components of the kit include a buffer solution, coenzyme, glutamic acid, adenyl pyrophosphate, glyceraldehydes-3-phosphoric acid, glutamoyl synthetase, glyceraldehydes-3-phosphate dehydrogenaseand a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, a series of enzymatic reactions occur, then the reactant is placed under an ultraviolet/visible light analyzer, and the degree/velocity of the increase in absorbance at 340 nm of the dominant wavelength is detected, thereby measuring the concentration of ammonia (ions).
Description
Technical field
The present invention relates to a kind of ammonia (ion) diagnosis/determination kit, the invention still further relates to the method for measuring ammonia (ion) 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, metering reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for ammonia (ion) concentration, simultaneously, the present invention also will provide ammonia (ion) diagnosis/determination kit in order to realize this method, adopt this reagent not only can be ultraviolet analyser or half, carry out ammonia (ion) concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Ammonia of the present invention (ion) method for measurement of concentration principle is as follows:
Ammonium ion+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+glyceraldehyde-3-phosphate+coenzyme
Glyceraldehyde-3-phosphate dehydrogenase
Glyceric acid-1,3-bis phosphoric acid+reduced coenzyme
This method is used glutamy and is pressed synzyme (glutamine synthetase; EC6.3.1.2) enzyme (idol) connection glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase; EC1.2.1.59) enzymatic reaction end-point method.Glutamy is pressed synzyme enzymolysis ammonia (ion) reaction and is produced phosphate radical, the effect of uniting glyceraldehyde-3-phosphate 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 degree that reduced coenzyme rises in 340nm place absorbance, by measuring the degree that 340nm place absorbance rises, can calculate the concentration of ammonia (ion).
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 ammonia of the present invention (ion) diagnosis/determination kit of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Glutamy is pressed synthetase 1 0000U/L
Glyceraldehyde-3-phosphate dehydrogenase 12000U/L
Glutamic acid 8mmol/L
Adenosine triphosphate 3mmol/L
Glyceraldehyde-3-phosphate 5mmol/L
Ammonia of the present invention (ion) diagnosis/determination kit can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, glutamy are pressed synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate.
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, glutamy are pressed synzyme, glyceraldehyde-3-phosphate dehydrogenase.
Coenzyme, glutamy can not limit by synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, the position of glyceraldehyde-3-phosphate in reagent 1 or reagent 2.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, adenosine triphosphate, glyceraldehyde-3-phosphate.
Reagent 2
Damping fluid, stabilizing agent, glyceraldehyde-3-phosphate dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, glutamy are pressed synzyme.
Coenzyme, glutamy can not limit by synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, the position of glyceraldehyde-3-phosphate in reagent 1, reagent 2 or reagent 3.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 (ion) 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 of present embodiment (ion) diagnosing/determining reagent is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Glutamy is pressed synthetase 1 0000U/L
Glyceraldehyde-3-phosphate dehydrogenase 12000U/L
Glutamic acid 8mmol/L
Adenosine triphosphate 3mmol/L
Glyceraldehyde-3-phosphate 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 ammonia (ion) sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), 0 minute 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 (ion).
Embodiment two
The ammonia of present embodiment (ion) diagnosing/determining reagent is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glutamic acid 8mmol/L
Adenosine triphosphate 3mmol/L
Glyceraldehyde-3-phosphate 5mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glutamy is pressed synthetase 1 0000U/L
Glyceraldehyde-3-phosphate dehydrogenase 12000U/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 (ion) sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), 0 minute 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 (ion).
Embodiment three
The ammonia of present embodiment (ion) diagnosing/determining reagent is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glutamic acid 8mmol/L
Adenosine triphosphate 3mmol/L
Glyceraldehyde-3-phosphate 5mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glyceraldehyde-3-phosphate dehydrogenase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutamy is pressed synthetase 1 0000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring ammonia (ion) 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 (ion) sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is positive reaction (reaction of rising), and 0 minute 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 (ion).
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)≤0.0015; Absorbance time response curve should be the rising curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 2mmol/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 that 0.68 ± 0.12 Δ A/mmol/L---the present invention is highly sensitive, degree of accuracy good, and the linear range broadness is enough to easy to utilize.
Claims (6)
1. the method for measurement of concentration of the ammonia (ion) of enzymic colorimetric and enzyme-linked method, its method principle is as follows:
Ammonium ion+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+glyceraldehyde-3-phosphate+coenzyme
Glyceraldehyde-3-phosphate dehydrogenase
Glyceric acid-1,3-bis phosphoric acid+reduced coenzyme
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 (ion).
2. an ammonia (ion) diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Glutamy is pressed synthetase 1 000---80000U/L
Glyceraldehyde-3-phosphate dehydrogenase 1000---80000U/L
Glutamic acid 1---50mmol/L
Adenosine triphosphate 1---50mmol/L
Glyceraldehyde-3-phosphate 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 ammonia of claim 2 (ion) diagnosis/determination kit, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, glutamy by synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate.
4. according to the described ammonia of claim 2 (ion) diagnosis/determination kit, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, glutamy by synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate; Reagent 2 is made up of by synzyme, glyceraldehyde-3-phosphate dehydrogenase damping fluid, stabilizing agent, glutamy.Coenzyme, glutamy can not limit by synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, the position of glyceraldehyde-3-phosphate in reagent 1 or reagent 2.
5. according to the described ammonia of claim 2 (ion) diagnosis/determination kit, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, glutamy by synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, glyceraldehyde-3-phosphate; 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, glyceraldehyde-3-phosphate dehydrogenase; Reagent 3 is made up of by synzyme damping fluid, stabilizing agent, glutamy.Coenzyme, glutamy can not limit by synzyme, glyceraldehyde-3-phosphate dehydrogenase, glutamic acid, adenosine triphosphate, the position of glyceraldehyde-3-phosphate in reagent 1, reagent 2 or reagent 3.
6. according to the described ammonia of claim 2 (ion) diagnosis/determination kit, 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 |
|---|---|---|---|
| CNA2007101921348A CN101464341A (en) | 2007-12-19 | 2007-12-19 | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101921348A CN101464341A (en) | 2007-12-19 | 2007-12-19 | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101464341A true CN101464341A (en) | 2009-06-24 |
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ID=40805017
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101921348A Pending CN101464341A (en) | 2007-12-19 | 2007-12-19 | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101464341A (en) |
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2007
- 2007-12-19 CN CNA2007101921348A patent/CN101464341A/en active Pending
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Open date: 20090624 |