CN101609019A - The method for measurement of concentration of isocitric acid measuring kit and isocitric acid - Google Patents
The method for measurement of concentration of isocitric acid measuring kit and isocitric acid Download PDFInfo
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- CN101609019A CN101609019A CNA2008101228325A CN200810122832A CN101609019A CN 101609019 A CN101609019 A CN 101609019A CN A2008101228325 A CNA2008101228325 A CN A2008101228325A CN 200810122832 A CN200810122832 A CN 200810122832A CN 101609019 A CN101609019 A CN 101609019A
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- isocitric
- isocitric acid
- stabilizing agent
- coenzyme
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Abstract
The present invention relates to a kind of isocitric acid measuring kit that utilizes the enzymic colorimetric technology, the invention still further relates to the composition and the composition of the reagent of measuring isocitric acid concentration simultaneously, belong to Food Inspection determination techniques field.Kit principal ingredient of the present invention comprises: damping fluid, coenzyme, isocitric dehydrogenase and stabilizing agent; By sample is mixed by certain volume ratio with reagent, make it to take place series of enzymatic reactions, again reactant is placed under the ultraviolet analyser, detect the degree that predominant wavelength 340nm place absorbance rises, thereby calculate the concentration of isocitric acid.This is that an efficient is very high, the method that cost is very cheap.Crucial the stabilizing agent that uses this law can make kit 2～8 ℃ preserve down stablized 1 year or more than, very easy to use.
Description
Technical field
The present invention relates to a kind of isocitric acid measuring kit, the invention still further relates to the method for measuring isocitric acid concentration simultaneously, belong to Food Inspection determination techniques field.
Background technology
Isocitric acid isocitric acid is the isomeride of citric acid, though amount is few, extensively is present in organic sphere.Many especially in the leaf of Bryophyllum succulent plants such as (Bryophyllum) or stirrup subclass, what biology can utilize is the D type.It is a composition in the tricarboxylic acid cycle.Citric acid reversibly generates isocitric acid and suitable achilleic acid under the effect of achilleic acid enzyme.At isocitric dehydrogenase (EC 1.1.1.41; EC 1.1.1.42) become α-Tong Wuersuan under the effect, (isocitrate lyase becomes succinic acid and glyoxalic acid under effect EC4.1.3.1) at isocitratase.
The State Standard of the People's Republic of China, GB T16771-1997 has stipulated the assay method-ultraviolet spectrophotometry of D-isocitric acid in orange, mandarin orange, orange juice and the beverage thereof.This method is applicable to judges orange, mandarin orange, tangerine inspissated juice and fruit juice, and juice content is not less than the mensuration of total D-isocitric acid of 2.5% orange, mandarin orange, orange juice beverage.Its measuring principle: isocitric dehydrogenase isocitrate dehydrogenase has two types: be the enzyme (EC1.1.1.41) of coenzyme and to want NADP be the enzyme (EC1.1.1.42) of coenzyme, the same reaction of both catalysis with NAD.
Under isocitric dehydrogenase catalysis, D-isocitrate in the sample and NAD or NADP effect generate the content of NADH or NADPH, are equivalent to the amount of D-isocitrate.Measure absorbance at wavelength 340nm place, determine the content of total D-isocitric acid in the sample.
Summary of the invention
The key technical problem that the present invention will solve is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) technology of utilizing, add suitable stabilizing agent, make the reagent can be under 28 ℃ of preservations, stablize more than 1 year, metering reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for isocitric acid concentration, simultaneously, the present invention also will provide in order to realize the isocitric acid measuring kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out isocitric acid concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, stability is high, easy to use, thereby can obtain practical applying.
Isocitric acid concentration determination method principle of the present invention is as follows:
Isocitric acid+coenzyme
Isocitric dehydrogenaseCarbon dioxide+ALPHA-KG+
Reduced coenzyme
This method is used isocitric dehydrogenase isocitrate dehydrogenase; EC 1.1.1.41; EC 1.1.1.42) enzymatic reaction end-point method.Isocitric dehydrogenase enzymolysis isocitric acid, 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 isocitric acid.
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 isocitric acid measuring kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Isocitric dehydrogenase 10000U/L
Isocitric acid measuring kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, isocitric 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.
Reagent 2
Damping fluid, stabilizing agent, isocitric dehydrogenase.
Coenzyme, the position of isocitric 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.
No matter be single agent or two agent, the present invention measures the method for isocitric 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
The isocitric acid determination reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Isocitric dehydrogenase 10000U/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 isocitric acid 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 isocitric acid.
Embodiment two
The isocitric acid determination reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Isocitric 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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested isocitric acid 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 isocitric acid.
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)≤0.002; 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 20mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 4%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.1 ± 0.05 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.This is that an efficient is very high, the method that cost is very cheap.
Claims (4)
1. isocitric acid measuring kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Isocitric dehydrogenase 1000---80000U/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.
2. according to the described isocitric acid measuring kit of claim 2, it is characterized in that: form single agent reagent by damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase.
3. according to the described isocitric acid measuring kit of claim 2, it is characterized in that: form two agent reagent by damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, isocitric dehydrogenase.Coenzyme, the position of isocitric dehydrogenase in reagent 1 or reagent 2 can not limit.
4. according to the described isocitric acid measuring 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 (AmmoniaSulfate), 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 |
|---|---|---|---|
| CNA2008101228325A CN101609019A (en) | 2008-06-19 | 2008-06-19 | The method for measurement of concentration of isocitric acid measuring kit and isocitric acid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101228325A CN101609019A (en) | 2008-06-19 | 2008-06-19 | The method for measurement of concentration of isocitric acid measuring kit and isocitric acid |
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| Publication Number | Publication Date |
|---|---|
| CN101609019A true CN101609019A (en) | 2009-12-23 |
Family
ID=41482848
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008101228325A Pending CN101609019A (en) | 2008-06-19 | 2008-06-19 | The method for measurement of concentration of isocitric acid measuring kit and isocitric acid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114908139A (en) * | 2022-07-15 | 2022-08-16 | 北京索莱宝科技有限公司 | Activity detection system of mitochondria isocitrate dehydrogenase and application thereof |
-
2008
- 2008-06-19 CN CNA2008101228325A patent/CN101609019A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114908139A (en) * | 2022-07-15 | 2022-08-16 | 北京索莱宝科技有限公司 | Activity detection system of mitochondria isocitrate dehydrogenase and application thereof |
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Open date: 20091223 |