CN101464308A - Isocitric acid measuring reagent kit and isocitric acid concentration determination method - Google Patents
Isocitric acid measuring reagent kit and isocitric acid concentration determination method Download PDFInfo
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- CN101464308A CN101464308A CNA2007101921013A CN200710192101A CN101464308A CN 101464308 A CN101464308 A CN 101464308A CN A2007101921013 A CNA2007101921013 A CN A2007101921013A CN 200710192101 A CN200710192101 A CN 200710192101A CN 101464308 A CN101464308 A CN 101464308A
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- isocitric
- dehydrogenase
- coenzyme
- ammonium ion
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Abstract
The invention relates to a kit for measuring isocitric acid by utilizing the technologies of the enzymatic cycling amplification method, 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 the isocitric acid, and belongs to the technical field of food inspection and measurement. The main components of the kit include a buffer solution, coenzyme, peroxide, ammonium ions, isocitrate dehydrogenase, dglutamic oxidase, glutamic 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 velocity of the increase in absorbance at 340 nm of the dominant wavelength is detected, thereby measuring the concentration of isocitric acid.
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 (EC1.1.1.41; 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 technical problem to be solved in the present invention is: propose a kind of enzyme cycle amplification method (EnzymaticRecycling Method) of utilizing, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, metering/continuous monitoring 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, thereby can obtain practical applying.
Isocitric acid concentration determination method principle of the present invention is as follows:
D-isocitric acid+coenzyme
Isocitric dehydrogenaseCarbon dioxide+
Ketoglutaric acid+reduced coenzyme
Ketoglutaric acid+ammonium ion+hydrogen peroxide
Dglutamic oxidaseL-glutamic acid+
Oxygen+water
L-glutamic acid+water+coenzyme
Glutamte dehydrogenaseAmmonium ion+ketoglutaric acid+
Reduced coenzyme
This method is used isocitric dehydrogenase (isocitrate dehydrogenase; EC 1.1.1.41; EC1.1.1.42) enzyme (idol) connection dglutamic oxidase (L-glutamate oxidase; EC 1.4.3.7; EC1.4.3.11), glutamte dehydrogenase (Glutamate dehydrogenase; EC 1.4.1.2; EC 1.4.1.3) enzyme ' s reaction speeding colourimetry.Isocitric dehydrogenase, as the effect enzyme, the reaction of enzymolysis isocitric acid produces ketoglutaric acid, also is simultaneously the colour developing enzyme, with coenzyme reduction becoming reduced coenzyme; By the effect of (idol) associating dglutamic oxidase, glutamte dehydrogenase, dglutamic oxidase utilizes ketoglutaric acid to produce glutamic acid also as the effect enzyme again; Glutamte dehydrogenase is again the colour developing enzyme as cyclophorase promptly, and one side becomes glutamic acid again ketoglutaric acid, makes it constantly recycling; One side is again with coenzyme (not having absorption peak at the 340nm place) reduction becoming reduced coenzyme (absorption peak being arranged 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 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
Dglutamic oxidase 12000U/L
Glutamte dehydrogenase 12000U/L
Hydrogen peroxide 10mmol/L
Ammonium ion 10mmol/L
Isocitric acid measuring kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, ammonium ion.
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.Ammonium ion can be any ammonia salt, as sal-ammoniac.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, ammonium ion.
Reagent 2
Damping fluid, stabilizing agent, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase.
Coenzyme, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, the position of ammonium ion 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, coenzyme, hydrogen peroxide, ammonium ion.
Reagent 2
Damping fluid, stabilizing agent, dglutamic oxidase, glutamte dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, isocitric dehydrogenase.
Coenzyme, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, the position of ammonium ion 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 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
Dglutamic oxidase 12000U/L
Glutamte dehydrogenase 12000U/L
Hydrogen peroxide 10mmol/L
Ammonium ion 10mmol/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 2 minutes of time delay is about 3 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 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
Hydrogen peroxide 10mmol/L
Ammonium ion 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Isocitric dehydrogenase 10000U/L
Dglutamic oxidase 12000U/L
Glutamte 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 isocitric acid sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), about about 2 minutes of time delay is about 3 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 the concentration of isocitric acid.
Embodiment three
The isocitric acid determination 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 10mmol/L
Ammonium ion 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Dglutamic oxidase 12000U/L
Glutamte dehydrogenase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Isocitric 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 isocitric 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 isocitric 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 2 minutes of time delay is about 3 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 the concentration of isocitric acid.
The applicant adopts the assay method 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 the rising curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 9.6mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤3.5% of the precision of reagent test (repeatability); The sensitivity of reagent can reach that 0.1022 ± 0.051 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 isocitric acid of an enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method, its method principle is as follows:
D-isocitric acid+coenzyme
Isocitric dehydrogenaseCarbon dioxide+
Ketoglutaric acid+reduced coenzyme
Ketoglutaric acid+ammonium ion+hydrogen peroxide
Dglutamic oxidaseL-glutamic acid+
Oxygen+water
L-glutamic acid+water+coenzyme
Glutamte dehydrogenaseAmmonium ion+ketoglutaric 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 the concentration measurement result of isocitric acid.
2. 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
Dglutamic oxidase 1000---80000U/L
Glutamte dehydrogenase 1000---80000U/L
Hydrogen peroxide 1---50mmol/L
Ammonium ion 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.Ammonium ion can be any ammonia salt, as sal-ammoniac.
3. 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, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, ammonium ion.
4. 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, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, ammonium ion; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, ammonium ion; Reagent 2 is made up of damping fluid, stabilizing agent, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase.Coenzyme, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, the position of ammonium ion in reagent 1 or reagent 2 can not limit.
5. according to the described isocitric acid measuring kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, ammonium ion; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, ammonium ion; Reagent 2 is made up of damping fluid, stabilizing agent, dglutamic oxidase, glutamte dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, isocitric dehydrogenase.Coenzyme, isocitric dehydrogenase, dglutamic oxidase, glutamte dehydrogenase, hydrogen peroxide, the position of ammonium ion in reagent 1, reagent 2 or reagent 3 can not limit.
6. 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 (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 |
|---|---|---|---|
| CNA2007101921013A CN101464308A (en) | 2007-12-19 | 2007-12-19 | Isocitric acid measuring reagent kit and isocitric acid concentration determination method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101921013A CN101464308A (en) | 2007-12-19 | 2007-12-19 | Isocitric acid measuring reagent kit and isocitric acid concentration determination method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101464308A true CN101464308A (en) | 2009-06-24 |
Family
ID=40804983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101921013A Pending CN101464308A (en) | 2007-12-19 | 2007-12-19 | Isocitric acid measuring reagent kit and isocitric acid concentration determination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101464308A (en) |
-
2007
- 2007-12-19 CN CNA2007101921013A patent/CN101464308A/en active Pending
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Open date: 20090624 |