CN101169408A - 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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- CN101169408A CN101169408A CNA2006100972244A CN200610097224A CN101169408A CN 101169408 A CN101169408 A CN 101169408A CN A2006100972244 A CNA2006100972244 A CN A2006100972244A CN 200610097224 A CN200610097224 A CN 200610097224A CN 101169408 A CN101169408 A CN 101169408A
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- reagent
- stabilizing agent
- ammonia
- damping fluid
- reduced coenzyme
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Abstract
The invention relates to an ammonia diagnosis/measuring reagent box utilizing the technology of enzyme colorimetry and an enzyme-linked method, and at the same time, the invention also relates to a method principle for measuring the ammonia concentration, and the compositions and ingredients of reagent, and belongs to the technical field of medical/food/environment checking and measuring. The reagent box of the invention mainly comprises buffer solution, pyruvic acid, alanine dehydrogenases, reduced coenzyme and stabilizing agent. The sample and the reagent are mixed according to a certain volume so as to generate a series of enzymatic reactions; and then the reactant is put under an ultraviolet/visible light analyzer to detect the decreasing speed of absorbance at the main wave length of 340 nanometers and calculate the ammonia concentration. By use of the ultraviolet/visible light analyzer, the invention can obtain the required measuring result.
Description
Technical field
The present invention relates to a kind of ammonia diagnosis/determination kit, the invention still further relates to the assay method of 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) technology of utilizing, metering 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 the mensuration of ammonia concentration 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+pyruvic acid+reduced coenzyme
Alanine dehydrogenaseAlanine+
Water+coenzyme
This method is used alanine dehydrogenase enzymatic reaction terminal colorimetric analysis.The effect of alanine dehydrogenase enzymolysis ammonia, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into oxidized coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured the degree that reduced coenzyme descends in 340nm place absorbance, by measuring the degree that 340nm place absorbance descends, can calculate the size of ammonia concentration.
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
Reduced coenzyme 0.25mmol/L
Pyruvic acid 12mmol/L
Alanine dehydrogenase 12000U/L
Ammonia diagnosis/determination kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, alanine 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, reduced coenzyme, pyruvic acid.
Reagent 2
Damping fluid, stabilizing agent, alanine dehydrogenase.
Reduced coenzyme, pyruvic acid, the position of alanine 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.
Also above-mentioned single agent reagent can be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme.
Reagent 2
Damping fluid, stabilizing agent, pyruvic acid.
Reagent 3
Damping fluid, stabilizing agent, alanine dehydrogenase.
Reduced coenzyme, pyruvic acid, the position of alanine 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 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 ammonia diagnosing/determining reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Pyruvic acid 12mmol/L
Alanine dehydrogenase 12000U/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 ammonia sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), detection method is an end-point method, 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 descends, thereby calculates the size of ammonia concentration.
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
Reduced coenzyme 0.25mmol/L
Pyruvic acid 16mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Alanine dehydrogenase 16000U/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 ammonia sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), detection method is an end-point method, 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 descends, thereby calculates the concentration of ammonia.
Embodiment three
The ammonia diagnosing/determining reagent of present embodiment is three reagent, comprising:
Reagent 1
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Reagent 2
Damping fluid 100mmol/L
Pyruvic acid 8mmol/L
Reagent 3
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Alanine dehydrogenase 8000U/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 1.8 ± 0.7, 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, and the Direction of Reaction is negative reaction (reaction descends), and detection method is an end-point method, 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 descends, thereby calculates the size of ammonia concentration.
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, be not subjected in the pollution of allogenic material, easy to utilize.
Claims (6)
1. ammonia concentration determination method, its method principle is as follows:
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the big or small degree that predominant wavelength 340nm absorbance descends, calculate ammonia concentration measurement result.
2. ammonia diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---50mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Pyruvic acid 1---50mmol/L
Alanine dehydrogenase 1000---80000U/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:
By damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, alanine dehydrogenase (alaninedehydrogenase; EC 1.4.1.1) forms single agent reagent.
4. according to the described ammonia diagnosis/determination kit of claim 2, it is characterized in that:
By damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, alanine dehydrogenase (alaninedehydrogenase; EC 1.4.1.1) forms two agent reagent; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, alanine dehydrogenase.Reduced coenzyme, pyruvic acid, the position of alanine 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:
By damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, alanine dehydrogenase (alaninedehydrogenase; EC 1.4.1.1) forms multi-agent reagent; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvic acid; Reagent 3 is made up of damping fluid, stabilizing agent, alanine dehydrogenase.Reduced coenzyme, pyruvic acid, the position of alanine 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 |
|---|---|---|---|
| CNA2006100972244A CN101169408A (en) | 2006-10-24 | 2006-10-24 | Ammonia diagnosis/determination reagent kit and ammonia concentration determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100972244A CN101169408A (en) | 2006-10-24 | 2006-10-24 | Ammonia diagnosis/determination reagent kit and ammonia concentration determination method |
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| Publication Number | Publication Date |
|---|---|
| CN101169408A true CN101169408A (en) | 2008-04-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006100972244A Pending CN101169408A (en) | 2006-10-24 | 2006-10-24 | Ammonia diagnosis/determination reagent kit and ammonia concentration determination method |
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| Country | Link |
|---|---|
| CN (1) | CN101169408A (en) |
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2006
- 2006-10-24 CN CNA2006100972244A patent/CN101169408A/en active Pending
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Open date: 20080430 |