CN101464350A - 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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- CN101464350A CN101464350A CNA2007101921437A CN200710192143A CN101464350A CN 101464350 A CN101464350 A CN 101464350A CN A2007101921437 A CNA2007101921437 A CN A2007101921437A CN 200710192143 A CN200710192143 A CN 200710192143A CN 101464350 A CN101464350 A CN 101464350A
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- acid
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- ammonia
- pyruvic acid
- stabilizing agent
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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, reduced coenzyme, glutamic acid, adenyl pyrophosphate, oxaloacetic acid, pyruvic acid, glutamine synthetase, phosphoric acid enol pyruvate carboxylase, malic dehydrogenase and 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 decrease 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, monitoring 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+oxaloacetic acid
Phosphoric acid enol pyruvic acid carboxylaseCarbon dioxide+
Phosphoenolpyruvic acid+water
Pyruvic acid+carbon dioxide+reduced coenzyme
Malic dehydrogenase (decarboxylation)
L MALIC ACID+coenzyme
This method is used glutamine synthelase (glutamine synthetase; EC 6.3.1.2) enzyme (idol) connection phosphoric acid enol pyruvic acid carboxylase (phosphoenolpyruvate carboxylase; EC 4.1.1.31), malic dehydrogenase (malate dehydrogenase; EC 1.1.1.38; EC 1.1.1.39; EC 1.1.1.40; EC 1.1.1.83) enzymatic reaction terminal colorimetric analysis.Glutamine synthelase enzymolysis ammonia (ion) reaction produces phosphate radical, the effect of uniting phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase 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 degree that reduced coenzyme descends in 340nm place absorbance, by measuring the degree that 340nm place absorbance descends, 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
Reduced coenzyme 0.25mmol/L
Glutamine synthelase 6000U/L
Phosphoric acid enol pyruvic acid carboxylase 8000U/L
Malic dehydrogenase 10000U/L
Glutamic acid 10mmol/L
Adenosine triphosphate 3mmol/L
Oxaloacetic acid 5mmol/L
Pyruvic acid 3mmol/L
Ammonia of the present invention (ion) diagnosis/determination kit can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid.
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, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid.
Reagent 2
Damping fluid, stabilizing agent, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase.
Reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, the position of pyruvic acid 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, reduced coenzyme, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid.
Reagent 2
Damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, glutamine synthelase.
Reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, the position of pyruvic acid 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 (ion) 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 of present embodiment (ion) diagnosing/determining reagent is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Glutamine synthelase 6000U/L
Phosphoric acid enol pyruvic acid carboxylase 8000U/L
Malic dehydrogenase 10000U/L
Glutamic acid 10mmol/L
Adenosine triphosphate 3mmol/L
Oxaloacetic acid 5mmol/L
Pyruvic acid 3mmol/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 (ion) sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), 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 descends, 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
Reduced coenzyme 0.25mmol/L
Glutamic acid 10mmol/L
Adenosine triphosphate 3mmol/L
Oxaloacetic acid 5mmol/L
Pyruvic acid 3mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutamine synthelase 6000U/L
Phosphoric acid enol pyruvic acid carboxylase 8000U/L
Malic dehydrogenase 10000U/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 (ion) sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), 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 descends, 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
Reduced coenzyme 0.25mmol/L
Glutamic acid 10mmol/L
Adenosine triphosphate 3mmol/L
Oxaloacetic acid 5mmol/L
Pyruvic acid 3mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Phosphoric acid enol pyruvic acid carboxylase 8000U/L
Malic dehydrogenase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutamine synthelase 6000U/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 1.8 ± 0.7, 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 negative reaction (reaction descends), 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 descends, 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)≤0.0012; Absorbance time response curve should be decline 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 0.63 ± 0.2 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. ammonia (ion) method for measurement of concentration that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Ammonium ion+glutamic acid+adenosine triphosphate
Glutamine synthelaseGlutamine+
Adenosine diphosphate+phosphate radical
Phosphate radical+oxaloacetic acid
Phosphoric acid enol pyruvic acid carboxylaseCarbon dioxide+
Phosphoenolpyruvic acid+water
Pyruvic acid+carbon dioxide+reduced coenzyme
Malic dehydrogenase (decarboxylation)
L MALIC ACID+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 descends, 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
Reduced coenzyme 0.1---0.35mmol/L
Glutamine synthelase 1000---80000U/L
Phosphoric acid enol pyruvic acid carboxylase 1000---80000U/L
Malic dehydrogenase 1000---80000U/L
Glutamic acid 1---50mmol/L
Adenosine triphosphate 1---50mmol/L
Oxaloacetic acid 1---50mmol/L
Pyruvic acid 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, reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid.
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, reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase.Reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, the position of pyruvic acid in reagent 1 or reagent 2 can not limit.
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, reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, glutamic acid, adenosine triphosphate, oxaloacetic acid, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, glutamine synthelase.Reduced coenzyme, glutamine synthelase, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, glutamic acid, adenosine triphosphate, oxaloacetic acid, the position of pyruvic acid in reagent 1, reagent 2 or reagent 3 can not limit.
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 (AmmoniaSulfate), 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 |
|---|---|---|---|
| CNA2007101921437A CN101464350A (en) | 2007-12-19 | 2007-12-19 | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101921437A CN101464350A (en) | 2007-12-19 | 2007-12-19 | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method |
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| CN101464350A true CN101464350A (en) | 2009-06-24 |
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| CNA2007101921437A Pending CN101464350A (en) | 2007-12-19 | 2007-12-19 | Ammonia ion diagnosis/measuring reagent kit and ammonia ion concentration determination method |
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Open date: 20090624 |