CN101750295A - Amino acid diagnosis/measurement reagent (kit) and amino acid concentration measurement method - Google Patents

Abstract

The invention relates to an amino acid diagnosis/measurement reagent (kit) utilizing an enzyme-colorimetry method and an enzyme immunoassay technology, and relates to a method for measuring the concentration of amino acid as well as the composition and the components of the reagent, belonging to the technical field of medical/food/environmental detection and measurement. The reagent (kit) mainly comprises the components of buffer solution, NADH, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine, amino acid oxidase, pyruvic oxidase, octopine dehydrogenase and a stabilizer. The concentration of amino acid is measured by the steps of: mixing a sample with the reagent in a certain volume radio to carry out a series of enzymatic reaction, placing the reactants under an ultraviolet/visible light analyzer, and detecting the reducing degree of absorbance at the main wavelength of 340 nm.

Description

Amino acid diagnosis/mensuration reagent (box) and amino acid concentration measurement method

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 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 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+oxygen Amino acid oxidaseOxoacid+ammonia+hydrogen peroxide

Hydrogen peroxide+carbon dioxide+acetyl phosphate Pyruvate oxidasePhosphate radical+

Pyruvic acid+oxygen+water

Pyruvic acid+arginine+reduced coenzyme Octopine dehydrogenase

N2-(D-1-carboxyethyl)-L-arginine+coenzyme+water

This method is used amino acid oxidase (amino-acid oxidase; EC 1.4.3.2; EC 1.4.3.3) enzyme (idol) connection pyruvate oxidase (pymvate oxidase; EC 1.2.3.3), octopine dehydrogenase (Octopinedehydrogenase; EC 1.5.1.11) enzymatic reaction colourimetry.The reaction of amino acid oxidase enzymolysis amino acid produces hydrogen peroxide, the effect of uniting pyruvate oxidase, octopine dehydrogenase 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 the degree that reduced coenzyme descends in 340nm place absorbance, by measuring the degree that 340nm place absorbance descends, 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

Reduced coenzyme 0.25mmol/L

Amino acid oxidase 6000U/L

Pyruvate oxidase 8000U/L

Octopine dehydrogenase 10000U/L

Sodium bicarbonate 6mmol/L

Acetyl phosphate 12mmol/L

Arginine 12mmol/L

Amino acid diagnosis/mensuration reagent of the present invention (box) can be single agent, comprising:

Damping fluid, stabilizing agent, reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine.

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, reduced coenzyme, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine.

Reagent 2

Damping fluid, stabilizing agent, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase.

Reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, the position of arginine 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, reduced coenzyme, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine.

Reagent 2

Damping fluid, stabilizing agent, pyruvate oxidase, octopine dehydrogenase.

Reagent 3

Damping fluid, stabilizing agent, amino acid oxidase.

Reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, the position of arginine 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 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

Amino 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

Amino acid oxidase 6000U/L

Pyruvate oxidase 8000U/L

Octopine dehydrogenase 10000U/L

Sodium bicarbonate 6mmol/L

Acetyl phosphate 12mmol/L

Arginine 12mmol/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 amino 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 the degree that predominant wavelength 340nm absorbance descends, 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

Reduced coenzyme 0.25mmol/L

Sodium bicarbonate 6mmol/L

Acetyl phosphate 12mmol/L

Arginine 12mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Amino acid oxidase 6000U/L

Pyruvate oxidase 8000U/L

Octopine 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 1.8 ± 0.7, 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 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 the degree that predominant wavelength 340nm absorbance descends, 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

Reduced coenzyme 0.25mmol/L

Sodium bicarbonate 6mmol/L

Acetyl phosphate 12mmol/L

Arginine 12mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Pyruvate oxidase 8000U/L

Grass carp alkali dehydrogenase 10 000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Amino acid oxidase 6000U/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 1.8 ± 0.7, 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 negative reaction (reaction descends), and 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 descends, 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 decline 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 4%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.0025 ± 0.0015TTT 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. amino acid concentration measurement method that utilizes enzymic colorimetric and enzyme-linked method technology, its method is as follows:

Amino acid+water+oxygen Amino acid oxidaseOxoacid+ammonia+hydrogen peroxide

Hydrogen peroxide+carbon dioxide+acetyl phosphate Pyruvate oxidasePhosphate radical+

Pyruvic acid+oxygen+water

Pyruvic acid+arginine+reduced coenzyme Octopine dehydrogenase

N2-(D-1-carboxyethyl)-L-arginine+coenzyme+water

The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbance descends, 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

Reduced coenzyme 0.1---0.35mmol/L

Amino acid oxidase 1000---80000U/L

Pyruvate oxidase 1000---80000U/L

Octopine dehydrogenase 1000---80000U/L

Sodium bicarbonate 1---50mmol/L

Acetyl phosphate 1---50mmol/L

Arginine 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, reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine.

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, reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine; Reagent 2 is made up of damping fluid, stabilizing agent, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase.Reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, the position of arginine 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, reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, sodium bicarbonate (carbon dioxide), acetyl phosphate, arginine; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate oxidase, octopine dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, amino acid oxidase.Reduced coenzyme, amino acid oxidase, pyruvate oxidase, octopine dehydrogenase, sodium bicarbonate (carbon dioxide), acetyl phosphate, the position of arginine 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: ammonium sulfate (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.