CN101762494A - Reagent (kit) for diagnosing/determining amino acid and method for determining concentration of amino acid - Google Patents
Reagent (kit) for diagnosing/determining amino acid and method for determining concentration of amino acid Download PDFInfo
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- CN101762494A CN101762494A CN200810235679A CN200810235679A CN101762494A CN 101762494 A CN101762494 A CN 101762494A CN 200810235679 A CN200810235679 A CN 200810235679A CN 200810235679 A CN200810235679 A CN 200810235679A CN 101762494 A CN101762494 A CN 101762494A
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- reagent
- amino acid
- coenzyme
- stabilizing agent
- hydrogen peroxide
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Abstract
The invention relates to a reagent (kit) for diagnosing/determining amino acid by utilizing the techniques of an enzymatic recycling method, an enzyme colorimetric method and an enzyme coupling method, and also relates to a method for determining the concentration of amino acid and a composition and components of the reagent, belonging to the technical field of detection and determination of medicine/foodstuff/environment. The reagent (kit) mainly comprises the following components: a buffer solution, coenzyme, hydrogen peroxide, 2-ketoglutarate, amino acid dehydrogenase, glutamate oxidase, glutamate synthetase and a stabilizer. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymic reactions, then a reactant is placed under an ultraviolet/visible light analyzer, and the rising speed of absorbance in the position of 340 nm of main wave length is detected so that the concentration of the amino acid is measured and calculated.
Description
Technical field
The present invention relates to a kind of amino acid diagnosis/mensuration reagent (box), the invention still further relates to the method for measuring amino acid concentration simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
Being determined in medical science/food/industry/agricultural/environment of amino acid content all is important mensuration project.Existing assay method has methods such as chromatography, electrochemical process, instrumental method (amino-acid analyzer), spectrophotometer, and these methods or method of operating are comparatively numerous and diverse, poor specificity or the more high shortcoming of instrument and equipment cost.
Amino acid is not simple a kind of material, (instrument costs an arm and a leg can directly to determine 17 seed amino acids with amino-acid analyzer, can not generally use), in medical science/food/industry/agricultural/environment, in most cases, all be that a variety of amino acid exist simultaneously, so need to measure total amino acid content, they can not be represented with the amino acid percent, can only represent with the percent of nitrogen contained in the amino acid (amino acid nitrogen).
Amino acid nitrogen increase in urine when a large amount of food meat or hunger, pregnant woman and neonatal urine amino acid nitrogen also increase.Amino acid metabolism is unusual, causes some amino acid to accumulate in vivo too much, makes that amino acid nitrogen increases in the urine; Acute liver atrophy, liver failure, Reye syndrome or some factor cause protein to decompose the disease of quickening, and the metabolic disorder that genetic disease caused all can make amino acid nitrogen increase in the urine.
Amino acid nitrogen content has specificity height, method is easy, cost is low characteristics in enzymatic assays blood, urine, body fluid, food, soil, the industrial products.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzyme cycle amplification method (EnzymaticRecycling Method) of utilizing, enzyme multiplication method (Enzymatic Doubling Method), enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, metering reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for amino acid concentration, simultaneously, the present invention also will provide in order to realize the amino acid diagnosis/mensuration reagent (box) of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out amino acid concentration measurement on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Amino acid concentration measurement method of the present invention is as follows:
Amino acid+water+coenzyme
Amino acid dehydrogenaseOxyacid+ammonia+reduced coenzyme
Ammonia+hydrogen peroxide+2-oxoglutaric acid
Dglutamic oxidaseGlutamic acid+oxygen+water
Glutamic acid+coenzyme
Glutamate synthetaseGlutamine+2-oxoglutaric acid+reduced coenzyme
This method is used amino acid dehydrogenase (amino-acid dehydrogenase; EC 1.4.1.5; EC1.4.99.1) enzyme (idol) connection dglutamic oxidase (glutamate oxidase; EC 1.4.3.7; EC 1.4.3.11; EC1.4.3.15), glutamate synthetase (glutamate synthase; EC 1.4.1.13) enzymatic reaction continuous monitoring method.Amino acid dehydrogenase is as the effect enzyme, and function is that the amino acid enzymolysis is produced ammonia.Dglutamic oxidase is as cyclophorase: the enzymatic catalysis of dglutamic oxidase makes ammonia become glutamic acid again, and glutamic acid on the one hand can be as the substrate of amino acid dehydrogenase, makes the interaction energy ringing again and again of amino acid dehydrogenase; Under the enzymatic catalysis of glutamate synthetase, glutamic acid also can be as the substrate of glutamate synthetase simultaneously.Amino acid dehydrogenase and glutamate synthetase are as the colour developing enzyme, with coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place 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 amino acid whose 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 amino acid diagnosis/mensuration reagent of the present invention (box) of following composition relation is ideal comparatively:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Amino acid dehydrogenase 10000U/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 12000U/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 16mmol/L
Amino acid diagnosis/mensuration reagent of the present invention (box) can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid.
Reagent (box) can be 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, hydrogen peroxide, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase.
Coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1 or reagent 2 can not limit.Reagent (box) can be 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, hydrogen peroxide, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, dglutamic oxidase, glutamate synthetase.
Reagent 3
Damping fluid, stabilizing agent, amino acid dehydrogenase.
Coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be 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 amino acid 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
Amino acid diagnosis/mensuration the reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Amino acid dehydrogenase 10000U/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 12000U/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 16mmol/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 amino acid sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay is about 2 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 speed that predominant wavelength 340nm absorbance rises, thereby calculates amino acid whose concentration.
Embodiment two
Amino acid diagnosis/mensuration the reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 16mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Amino acid dehydrogenase 10000U/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 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 amino acid 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 is about 2 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 speed that predominant wavelength 340nm absorbance rises, thereby calculates amino acid whose concentration.
Embodiment three
Amino acid diagnosis/mensuration the reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 16mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Amino acid dehydrogenase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring amino acid 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 amino acid 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 1 minute of time delay is about 2 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 speed that predominant wavelength 340nm absorbance rises, thereby calculates amino acid whose concentration.
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)≤0.0008; 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 40mmol/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 0.04 ± 0.02 Δ A/mmol/L; 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. the amino acid whose method for measurement of concentration of an enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method, its method is as follows:
Amino acid+water+coenzyme
Amino acid dehydrogenaseOxyacid+ammonia+reduced coenzyme
Ammonia+hydrogen peroxide+2-oxoglutaric acid
Dglutamic oxidaseGlutamic acid+oxygen+water
Glutamic acid+coenzyme
Glutamate synthetaseGlutamine+2-oxoglutaric 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 amino acid whose concentration measurement result.
2. an amino acid diagnosis/mensuration reagent (box), principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Amino acid dehydrogenase 1000---80000U/L
Dglutamic oxidase 1000---80000U/L
Glutamate synthetase 1000---80000U/L
Hydrogen peroxide 1---50mmol/L
2-oxoglutaric 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: reagent (box) can be 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 amino acid diagnosis/mensuration of claim 2 reagent (box), it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid.
4. according to the described amino acid diagnosis/mensuration of claim 2 reagent (box), it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase.Coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1 or reagent 2 can not limit.
5. according to the described amino acid diagnosis/mensuration of claim 2 reagent (box), it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, dglutamic oxidase, glutamate synthetase; Reagent 3 is made up of damping fluid, stabilizing agent, amino acid dehydrogenase.Coenzyme, amino acid dehydrogenase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described amino acid diagnosis/mensuration of claim 2 reagent (box), it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: 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 |
|---|---|---|---|
| CN200810235679A CN101762494A (en) | 2008-12-10 | 2008-12-10 | Reagent (kit) for diagnosing/determining amino acid and method for determining concentration of amino acid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810235679A CN101762494A (en) | 2008-12-10 | 2008-12-10 | Reagent (kit) for diagnosing/determining amino acid and method for determining concentration of amino acid |
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| Publication Number | Publication Date |
|---|---|
| CN101762494A true CN101762494A (en) | 2010-06-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810235679A Pending CN101762494A (en) | 2008-12-10 | 2008-12-10 | Reagent (kit) for diagnosing/determining amino acid and method for determining concentration of amino acid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104677905A (en) * | 2015-03-09 | 2015-06-03 | 大连工业大学 | Method for determining concentration of substrate of enzymatic reaction by detecting concentration of amino acid in reaction liquid |
-
2008
- 2008-12-10 CN CN200810235679A patent/CN101762494A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104677905A (en) * | 2015-03-09 | 2015-06-03 | 大连工业大学 | Method for determining concentration of substrate of enzymatic reaction by detecting concentration of amino acid in reaction liquid |
| CN104677905B (en) * | 2015-03-09 | 2017-03-08 | 大连工业大学 | A kind of method that amino acid concentration determines enzymatic reaction concentration of substrate in reactant liquor by detection |
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| C06 | Publication | ||
| PB01 | Publication | ||
| DD01 | Delivery of document by public notice |
Addressee: Aijie Biological Science & Technology Co., Ltd., Suzhou City Document name: Notification of Publication of the Application for Invention |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20100630 |