CN101793705A

CN101793705A - Isocitric acid determination reagent (kit) and isocitric acid concentration determination method

Info

Publication number: CN101793705A
Application number: CN200910028217A
Authority: CN
Inventors: 王尔中
Original assignee: Suzhou ANJ Biotech Co Ltd
Current assignee: Suzhou ANJ Biotech Co Ltd
Priority date: 2009-02-04
Filing date: 2009-02-04
Publication date: 2010-08-04

Abstract

The invention relates to an isocitric acid determination reagent (kit) which uses an enzyme multiplication method, an enzyme colorimetric method and an ELISA technology, and simultaneously relates to an isocitric acid concentration determination method as well as the composition and the components of the reagent, which belong to the technical field of food inspection and determination. The main components of the reagent (kit) of the invention comprise: buffer solution, coenzyme, acetyl coenzyme A, peroxide, glutathione, isocitrate dehydrogenase, pyruvate oxidase, coenzyme A-glutathione reductase and stabilizer; a sample is mixed with the reagent in a certain volume ratio, so that the sample and the reagent carry out a series of enzymatic reactions; and then the reactant is put under an UV/visible light analyzer, the increase of light absorbency at 340nm of a main wave length is detected, so as to measure the concentration of isocitric acid.

Description

Isocitric acid determination reagent (box) and isocitric acid concentration determination method

Technical field

The present invention relates to a kind of isocitric acid determination reagent (box), 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 technical problem to be solved in the present invention is: propose a kind of enzyme multiplication method (Enzymatic DoublingMethod) that utilizes, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (CoupleReaction) 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 determination reagent (box) 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 of the present invention is as follows:

Isocitric acid+coenzyme Isocitric dehydrogenaseCarbon dioxide+ketoglutaric acid+reduced coenzyme

Carbon dioxide+hydrogen peroxide+acetyl coenzyme A Pyruvate oxidasePyruvic acid+coacetylase

+ oxygen

Coacetylase+glutathione+coenzyme Coacetylase-glutathione reductaseCoacetylase-glutathione

+ reduced coenzyme

This method is used isocitric dehydrogenase (isocitrate dehydrogenase; EC 1.1.1.41; EC1.1.1.42) enzyme (idol) connection pyruvate oxidase (pyruvate oxidase; EC 1.2.3.6), coacetylase-glutathione reductase (CoA-glutathione reductase; EC 1.8.1.10) enzymatic reaction colorimetric end-point method.The reaction of isocitric dehydrogenase enzymolysis isocitric acid produces carbon dioxide, the effect of uniting pyruvate oxidase, coacetylase-glutathione reductase again by (idol), final secondary is 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 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 determination reagent of the present invention (box) of following composition relation is comparatively desirable:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Coenzyme 3mmol/L

Isocitric dehydrogenase 10000U/L

Pyruvate oxidase 12000U/L

Coacetylase-glutathione reductase 12000U/L

Acetyl coenzyme A 7mmol/L

Hydrogen peroxide 12mmol/L

Glutathione 3mmol/L

Isocitric acid determination reagent of the present invention (box) can be single agent, comprising:

Damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, glutathione.

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, acetyl coenzyme A, hydrogen peroxide, glutathione.

Reagent 2

Damping fluid, stabilizing agent, isocitric dehydrogenase, pyruvate oxidase, coacetylase-paddy Guang are sweet

Fabk polypeptide.

Coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, the position of glutathione 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

Reagent 2

Damping fluid, stabilizing agent, pyruvate oxidase, coacetylase-glutathione reductase.

Reagent 3

Damping fluid, stabilizing agent, isocitric dehydrogenase.

Coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, the position of glutathione 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 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

Pyruvate oxidase 12000U/L

Coacetylase-glutathione reductase 12000U/L

Acetyl coenzyme A 7mmol/L

Hydrogen peroxide 12mmol/L

Glutathione 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≤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

Acetyl coenzyme A 7mmol/L

Hydrogen peroxide 12mmol/L

Glutathione 3mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Isocitric dehydrogenase 10000U/L

Pyruvate oxidase 12000U/L

Coacetylase-glutathione reductase 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 0 minute of time delay is about 5 minutes detection times.

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

Acetyl coenzyme A 7mmol/L

Hydrogen peroxide 12mmol/L

Glutathione 3mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Pyruvate oxidase 12000U/L

Coacetylase-glutathione reductase 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 0 minute of time delay is about 5 minutes detection times.

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)≤0.0009; 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 3%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.024 ± 0.012 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

1. the method for measurement of concentration of the isocitric acid of an enzyme multiplication method, enzymic colorimetric and enzyme-linked method, its method is as follows:

+ oxygen

+ reduced 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 rises, calculate the concentration measurement result of isocitric acid.

2. an isocitric acid determination reagent (box), principal ingredient comprises:

Damping fluid 20---500mmol/L

Stabilizing agent 1---4000mmol/L

DPN diphosphopyridine nucleotide---6mmol/L

Isocitric dehydrogenase 1000---80000U/L

Pyruvate oxidase 1000---80000U/L

Coacetylase-glutathione reductase 1000---80000U/L

Acetyl coenzyme A 1---50mmol/L

Hydrogen peroxide 1---50mmol/L

Glutathione 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 isocitric acid determination reagent of claim 2 (box), it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, glutathione.

4. according to the described isocitric acid determination reagent of claim 2 (box), it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, glutathione; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, acetyl coenzyme A, hydrogen peroxide, glutathione; Reagent 2 is made up of damping fluid, stabilizing agent, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase.Coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, the position of glutathione in reagent 1 or reagent 2 can not limit.

5. according to the described isocitric acid determination reagent of claim 2 (box), it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, glutathione; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, acetyl coenzyme A, hydrogen peroxide, glutathione; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate oxidase, coacetylase-glutathione reductase; Reagent 3 is made up of damping fluid, stabilizing agent, isocitric dehydrogenase.Coenzyme, isocitric dehydrogenase, pyruvate oxidase, coacetylase-glutathione reductase, acetyl coenzyme A, hydrogen peroxide, the position of glutathione in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described isocitric acid determination reagent of claim 2 (box), 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.