CN101324585A - Method for determining amino acid (nitrogen) concentration and amino acid (nitrogen) diagnosis/determination reagent kit - Google Patents

Abstract

The invention relates to a kit for diagnosing/mensurating amino acid (nitrogen) by utilizing the technologies of the enzymatic cycling amplification method, 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 amino acid (nitrogen), and belongs to the technology field of medical/food/industrial/agricultural/environmental inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, glycine oxidase 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 amino acid (nitrogen).

Description

Amino acid (nitrogen) diagnosis/determination kit and amino acid (nitrogen) method for measurement of concentration

Technical field

The present invention relates to a seed amino acid (nitrogen) diagnosis/determination kit, the invention still further relates to the method for measuring amino acid (nitrogen) concentration simultaneously, belong to medical science/food/industry/agricultural/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, R e y e 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 enzyme cycle amplification method (Enzymatic Recycling Method) of utilizing, enzymic colorimetric (Enzymatic ColorimetricMethod) and enzyme (even) united method (Couple Reaction) technology, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for amino acid (nitrogen) concentration, simultaneously, the present invention also will provide amino acid (nitrogen) diagnosis/determination kit in order to realize this method, adopt this reagent not only can be ultraviolet analyser or half, carry out amino acid (nitrogen) concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Amino acid of the present invention (nitrogen) method for measurement of concentration principle is as follows:

Amino acid+water+oxygen Amino acid oxidaseOxoacid+ammonium ion+

Hydrogen peroxide

Ammonium ion+pyruvic acid+reduced coenzyme Alanine dehydrogenaseThe L-alanine

+ water+coenzyme

Alanine+water+oxygen Glycine oxidaseAmmonium ion+pyruvic acid+

Hydrogen peroxide

This method is used amino acid oxidase (amino acid oxidase; EC 1.4.3.2; EC1.4.3.3) enzyme (idol) connection alanine dehydrogenase (alanine dehydrogenase; EC 1.4.1.1), glycine oxidase (glycine oxidase; EC 1.4.3.19) enzymatic reaction continuous monitoring method.Amino acid oxidase is as the effect enzyme, and function is that the amino acid enzymolysis is produced ammonium ion.First enzymatic catalysis of alanine dehydrogenase makes ammonium ion produce alanine; Glycine oxidase can become alanine again ammonium ion again and again once more as cyclophorase, and it is recycling that ammonium ion constantly is repeated.Alanine dehydrogenase can be used as the colour developing enzyme again, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into oxidized coenzyme (not having absorption peak at the 340nm place), thereby measured speed/speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the concentration of amino acid (nitrogen).

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 amino acid of the present invention (nitrogen) diagnosis/determination kit of following composition relation is comparatively desirable:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvic acid 10mmol/L

Amino acid oxidase 10000U/L

Alanine dehydrogenase 10000U/L

Glycine oxidase 10000U/L

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

Damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, glycine oxidase.

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, pyruvic acid.

Reagent 2

Damping fluid, stabilizing agent, amino acid oxidase, alanine dehydrogenase, glycine oxidase.

Reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, the position of glycine oxidase 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, pyruvic acid.

Reagent 2

Damping fluid, stabilizing agent.Alanine dehydrogenase, glycine oxidase.

Reagent 3

Damping fluid, stabilizing agent, amino acid oxidase.

Reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, the position of glycine oxidase 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 amino acid (nitrogen) 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

The amino acid of present embodiment (nitrogen) diagnosing/determining reagent is single reagent, comprising:

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

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvic acid 10mmol/L

Amino acid oxidase 10000U/L

Alanine dehydrogenase 10000U/L

Glycine oxidase 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 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 degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of amino acid (nitrogen).

Embodiment two

The amino acid of present embodiment (nitrogen) diagnosing/determining reagent is double reagent, comprising:

Reagent 1

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

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvic acid 10mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Amino acid oxidase 10000U/L

Alanine dehydrogenase 10000U/L

Glycine oxidase 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 degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of amino acid (nitrogen).

Embodiment three

The amino acid of present embodiment (nitrogen) diagnosing/determining reagent is three reagent, comprising:

Reagent 1

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

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvic acid 10mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Alanine dehydrogenase 10000U/L

Glycine oxidase 10000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Amino acid 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 amino acid (nitrogen) 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 degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of amino acid (nitrogen).

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 (6)

1. amino acid (nitrogen) method for measurement of concentration that utilizes enzyme cycle amplification method, enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:

Amino acid+water+oxygen Amino acid oxidaseOxoacid+ammonium ion+

Hydrogen peroxide

Ammonium ion+pyruvic acid+reduced coenzyme Alanine dehydrogenaseThe L-alanine

+ water+coenzyme

Alanine+water+oxygen Glycine oxidaseAmmonium ion+pyruvic acid+

Hydrogen peroxide

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 amino acid (nitrogen).

2. a seed amino acid (nitrogen) diagnosis/determination kit, principal ingredient comprises:

Damping fluid 20-500mmol/L

Stabilizing agent 1-4000mmol/L

Reduced coenzyme 0.1-0.35mmol/L

Pyruvic acid 1-50mmol/L

Amino acid oxidase 1000-80000U/L

Alanine dehydrogenase 1000-80000U/L

Glycine oxidase 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 amino acid of claim 2 (nitrogen) diagnosis/determination kit, it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, glycine oxidase.

4. according to the described amino acid of claim 2 (nitrogen) diagnosis/determination kit, it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, glycine oxidase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, amino acid oxidase, alanine dehydrogenase, glycine oxidase.Reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, the position of glycine oxidase in reagent 1 or reagent 2 can not limit.

5. according to the described amino acid of claim 2 (nitrogen) diagnosis/determination kit, it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, glycine oxidase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, alanine dehydrogenase, glycine oxidase; Reagent 3 is made up of damping fluid, stabilizing agent, amino acid oxidase.Reduced coenzyme, pyruvic acid, amino acid oxidase, alanine dehydrogenase, the position of glycine oxidase in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described amino acid of claim 2 (nitrogen) diagnosis/determination 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 (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.