CN101324569A

CN101324569A - Glutamic acid diagnosis/determination reagent kit and method for determining glutamic acid concentration

Info

Publication number: CN101324569A
Application number: CNA2007100233923A
Authority: CN
Inventors: 王尔中
Original assignee: Suzhou ANJ Biotech Co Ltd
Current assignee: Suzhou ANJ Biotech Co Ltd
Priority date: 2007-06-13
Filing date: 2007-06-13
Publication date: 2008-12-17

Abstract

The invention relates to a kit for diagnosing/mensurating glutamic acid by utilizing the technologies of the enzymic colorimetric method and the enzyme linked immunosorbent assay (ELISA). The invention further relates to a method, a principle and the composition and the components of a reagent for mensurating the concentration of the glutamic acid, and belongs to the technology field of medical/food inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, glutamate oxidase, NADH peroxidase 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 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the glutamic acid.

Description

Glutamic acid diagnosis/mensuration kit and aminoglutaric acid concentration assay method

Technical field

The present invention relates to a kind of glutamic acid diagnosis/mensuration kit, the invention still further relates to the method for measuring aminoglutaric acid concentration simultaneously, belong to medical science/Food Inspection determination techniques field.

Background technology

State Standard of the People's Republic of China GB5009-2003 monosodium glutamate hygienic standard analytical approach has: 1. polarimeter method 2. formol titration 3. Kjeldahls.Monosodium glutamate means that with grain and goods be the sodium glutamate that raw material is purified through fermentation.

Enzymatic analysis is the analytical approach of the glutamate of generally acknowledging in the world, organized by a plurality of internal authorities or mechanism adopts: as IFU (international fruit juice association of producers), AIJN (European Union's Juice and beverage production person alliance), and MEBAK, OICC, VDLUFA etc. organize widely and praise highly, and are defined as standard detecting method.Be written into Australia, Holland, Germany, the food law of a plurality of countries such as Switzerland.Domestic also have the man fruit juice of number, drinks processing enterprise to use the enzyme process kit as examination criteria.

Have osteoclast that decomposes sclerotin and the Gegenbaur's cell that forms bone in the biosome, the effect of the following two kinds of cells of normal condition is keeping good balance.If balance is broken, osteoclast is too active, will cause osteoporosis.Contain glutamic acid in the osteoclast, and the albumen of a kind of " glutamate transporter " by name can be discharged glutamic acid osteoclast, the glutamic acid meeting that is " released " and the receptors bind of cell surface, the enzyme in the active cell suppresses the effect that osteoclast decomposes sclerotin.

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 aminoglutaric acid concentration, simultaneously, the present invention also will provide in order to realize the glutamic acid diagnosis/mensuration kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out aminoglutaric acid concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Aminoglutaric acid concentration assay method principle of the present invention is as follows:

L-glutamic acid+oxygen+water Dglutamic oxidaseAmmonium ion+2-oxoglutaric acid ester

+ hydrogen peroxide

Hydrogen peroxide+reduced coenzyme The NADH peroxidaseCoenzyme+2 water

This method is used dglutamic oxidase (glutamate oxidase; EC 1.4.3.7; EC 1.4.3.11) coupling NADH peroxidase (NADH peroxidase; EC 1.11.1.1; EC 1.11.1.2) enzymatic reaction continuous monitoring method/terminal colorimetric analysis.The reaction of dglutamic oxidase enzymolysis glutamic acid produces hydrogen peroxide, the effect of uniting the NADH peroxidase 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 degree/speed that reduced coenzyme descends in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance descends, can calculate the concentration of glutamic 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 glutamic acid diagnosis/mensuration kit of the present invention of following composition relation is ideal comparatively:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Dglutamic oxidase 10000U/L

NADH peroxidase 16000U/L

Glutamic acid diagnosis/mensuration kit of the present invention can be single agent, comprising:

Damping fluid, stabilizing agent, reduced coenzyme, dglutamic oxidase, NADH peroxidase.

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.

Reagent 2

Damping fluid, stabilizing agent, dglutamic oxidase, NADH peroxidase.

Reduced coenzyme, dglutamic oxidase, the position of NADH peroxidase 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.

Reagent 2

Damping fluid, stabilizing agent, NADH peroxidase.

Reagent 3

Damping fluid, stabilizing agent, dglutamic oxidase.

Reduced coenzyme, dglutamic oxidase, the position of NADH peroxidase 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 aminoglutaric acid 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

Glutamic acid diagnosis/mensuration the reagent of present embodiment is single reagent, comprising:

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

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Dglutamic oxidase 10000U/L

NADH peroxidase 16000U/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 glutamic acid sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), 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 degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of glutamic acid.

Embodiment two

Glutamic 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

Reduced coenzyme 0.25mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Dglutamic oxidase 10000U/L

NADH peroxidase 16000U/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 glutamic acid sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 0 minute of time delay is about 5 minutes detection times.

Embodiment three

Glutamic 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

Reduced coenzyme 0.25mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

NADH peroxidase 16000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Dglutamic oxidase 10000U/L

Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.

When measuring aminoglutaric acid 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 glutamic acid sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), and about about 0 minute of time delay is about 5 minutes detection times.

The applicant adopts other various reduced form chromogens combinations 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, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, and is easy to utilize.

Claims

1. aminoglutaric acid concentration assay method that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:

+ hydrogen peroxide

Hydrogen peroxide+reduced coenzyme The NADH peroxidaseCoenzyme+2 water

The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance descends, calculate the concentration measurement result of glutamic acid.

2. glutamic acid diagnosis/mensuration kit, principal ingredient comprises:

Damping fluid 20-500mmol/L

Stabilizing agent 1-4000mmol/L

Reduced coenzyme 0.1-0.35mmol/L

Dglutamic oxidase 1000-80000U/L

NADH peroxidase 1000-80000U/L

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 glutamic acid diagnosis/mensuration of claim 2 kit, it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, dglutamic oxidase, NADH peroxidase.

4. according to the described glutamic acid diagnosis/mensuration of claim 2 kit, it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, dglutamic oxidase, NADH peroxidase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, dglutamic oxidase, NADH peroxidase.Reduced coenzyme, dglutamic oxidase, the position of NADH peroxidase in reagent 1 or reagent 2 can not limit.

5. according to the described glutamic acid diagnosis/mensuration of claim 2 kit, it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, dglutamic oxidase, NADH peroxidase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, NADH peroxidase; Reagent 3 is made up of damping fluid, stabilizing agent, dglutamic oxidase.Reduced coenzyme, dglutamic oxidase, the position of NADH peroxidase in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described glutamic acid diagnosis/mensuration of claim 2 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 (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.