CN102298011A - Method for determining glycine and glycine determination kit - Google Patents

Abstract

The invention relates to a method for determining the glycine content by utilizing a double amplification method, an enzymatic colorimetric method and couple reaction as well as composition and components of a reagent. The technology principle of the method is as follow: determination is completed based on serial catalytic reactions of hydrogen cyanide synthase, pyruvate oxidase and glyceraldehyde-3-phosphate dehydrogenase. The invention also relates to a glycine determination kit. The method has the advantages of high sensitivity and few errors; and therefore the method and the kit can be widely applied to food inspection.

Description

The assay method of glycocoll and glycocoll are measured kit

[technical field]

The present invention relates to Food Inspection determination techniques field, more specifically, the present invention relates to glycocoll assay method and kit thereof.

[background technology]

Glycocoll is non-essential amino acid.Glycocoll has unique sweet taste, can relax acid, alkali flavor, covers and adds the bitter taste of asccharin in the food and strengthen sweet taste.Human body is too much if take in the amount of glycocoll, and the utilization that not only can not be absorbed by the body, and can break human body amino acid whose absorption equilibrium is influenced other amino acid whose absorption causes nutrient imbalance and unhealthful.Glycocoll can not be utilized by human body, can only decomposition and inversion be heat energy, can increase burden of liver like this.And the nitrogen-containing compound in the decomposition product will excrete by urine, can increase the kidney burden again.If eat this based food in a large number, for a long time, may cause lesions of liver and kidney.Part milk-contained drink enterprise uses glycocoll in violation of rules and regulations for improving protein content in the product in production and processing, teenager and children's normal growth is grown be easy to bring adverse effect.

According to China's " food additives use hygienic standard " GB2760 regulation, glycocoll is only allowing to use in flavoring additives and soymilk as food additives, and the highest limiting the quantity of be per kilogram 1 gram, but does not allow use in milk-contained drink.

[summary of the invention]

[technical matters that will solve]

The assay method that the purpose of this invention is to provide a kind of glycocoll.

Another object of the present invention provides a kind of glycocoll and measures kit.

[technical scheme]

The present invention is achieved through the following technical solutions.

Method of the present invention is a kind of coupling technique that adopts two times of TRAP, enzymic colorimetric (Enzymatic ColorimetricMethod) and enzyme (even) united method (Couple Reaction), utilizes reduced form nicotinamide coenzyme (reduced coenzyme) is measured glycocoll in the absorbance variation at 340nm wavelength place the method for measuring.

Glycocoll assay method of the present invention know-why be to finish according to the serial catalytic reaction of following hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase:

Glycocoll+2 coenzyme Hydrogen cyanide synthaseHydrogen cyanide+carbon dioxide+2 reduced coenzymes

Carbon dioxide+acetyl phosphate+hydrogen peroxide Pyruvate oxidasePhosphate radical+pyruvic acid

+ oxygen+water

Phosphate radical+glyceraldehyde-3-phosphate+coenzyme Glyceraldehyde-3-phosphate dehydrogenase

Glyceric acid-1,3-bis phosphoric acid+reduced coenzyme

Method of the present invention is utilized hydrogen cyanide synthase (hydrogen cyanide synthase; EC 1.4.99.5) enzyme (idol) connection pyruvate oxidase (pyruvate oxidase; EC 1.2.3.3), glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase; EC 1.2.1.59) enzymatic reaction end-point method.Hydrogen cyanide synthase enzymolysis glycine reactant produces carbon dioxide, the effect of uniting pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase again by (idol), coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured the degree that reduced coenzyme rises in 340nm place absorbance, can can calculate the concentration of glycocoll by measuring the degree that 340nm place absorbance rises like this.

The present invention is achieved through the following technical solutions.

The present invention relates to a kind of assay method of glycocoll.The step of this glycocoll assay method is as follows:

A, sample are prepared:

A.1 the preparation of standard model

With a certain amount of glycocoll in the water-soluble or damping fluid, again glycine concentration is adjusted to 1 mM/liter, the solution that obtains is as standard model;

A.2 the preparation of testing sample

The testing liquid sample is directly tested, need not pre-service; With a certain amount of solid sample to be measured as the preparation standard sample in the water-soluble or damping fluid;

A.3 blank sample

Described water or damping fluid be as blank sample, its glycine concentration be 0 mM/liter;

The preparation of B, reagent solution:

Pipette or take by weighing damping fluid, stabilizing agent, coenzyme, hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase, acetyl phosphate, hydrogen peroxide and glyceraldehyde-3-phosphate respectively, then they are mixed, the water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-5mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/L, 1-100mmol/L, 1-100mmol/L and 1-100mmol/L;

C, testing sample with at step B) reagent solution that obtains mixes according to volume ratio 1/10 to 1/500, reacted 5-60 minute down at temperature 15-45 ℃, under predominant wavelength 340nm and commplementary wave length 405nm (can not establish commplementary wave length), measure, measure its absorbance over time if be subjected to the instrument restriction;

D, with step C) determination step A under the same condition) and standard model absorbance over time;

E, with step C) be determined at steps A under the same condition) absorbance of blank sample over time;

F, data processing

By step C-E) absorbance of the predominant wavelength 340nm of described mensuration over time, calculate the content of glycocoll according to following formula:

In the formula:

Δ A (sample) expression step C) absorbance of the testing sample that obtains changes;

Δ A (blank) represents step e) absorbance of the blank sample that obtains changes;

Δ A (standard) expression step D) absorbance of standard model changes.

According to the present invention, in described glycocoll assay method, described damping fluid should be appreciated that it is to make it measure the solution of the pH kept stable (generally being 6.5-8.5) of medium.If this pH be higher than 8.5 or pH be lower than 6.5, then the activity of the enzyme that uses of this assay method does not reach the active effect of expection, therefore needs to add more medium and enzyme, just might reach the active effect of expection.

In the present invention, described damping fluid is one or more damping fluids that are selected from three (ethyloic) aminomethane-hydrochloride buffer, phosphate buffer, imidazoles-hydrochloride buffer, sodium hydrogen phosphate-citrate buffer solution, borax-hydrochloride buffer, barbital sodium-hydrochloride buffer, boric acid-borate buffer solution, diethanolamine buffer or " PBS " damping fluid.

Preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphate buffer or " PBS " damping fluid.

More preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid or phosphate buffer.

According to the present invention; in described glycocoll assay method; described stabilizing agent should be appreciated that it is a kind of medium (substrate) and the enzyme that can protect in the reagent; make it can not change its character as time passes; and then deactivated material; it can make reagent possess very long active lifetime, reaches the several months usually, even one, two year.If there is not described stabilizing agent, then the activity of reagent can only be kept tens of hours in solution, a couple of days at most, will lose activity gradually and no longer possessed the detection performance.In the present invention, described stabilizing agent use amount is 0.01-7mol/L.If described stabilizing agent use amount is not enough, then the life-span of agent of activity will shorten; If described stabilizing agent use amount is too high, then can increase cost.

In the present invention, described stabilizing agent is one or more stabilizing agents that are selected from ammonium sulfate, sodium chloride, ethylene glycol, propylene glycol, glycerine or sodium Diacetate or Sodium azide antiseptic.

Preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from ammonium sulfate, sodium chloride, propylene glycol, glycerine or sodium Diacetate or Sodium azide.

More preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from glycerine, ammonium sulfate or sodium Diacetate.

In the present invention, described coenzyme is that one or more are selected from NADP ⁺, NAD ⁺Or thio-NAD ⁺Coenzyme.

According to a kind of preferred implementation of the present invention, when the present invention uses automatic clinical chemistry analyzer to measure glycocoll, testing sample, described standard model and described blank sample are by (parameter) sampling automatically under imposing a condition of this analyser, measure under the following conditions then: assay method is 2 end-point method/end-point methods, 37 ℃ of temperature, 10 minutes reaction time, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glycocoll sample and reagent is 1/10-1/500, the Direction of Reaction is positive reaction, 1/0 minute time delay, 4/5 minute detection time.

According to another kind preferred implementation of the present invention, the reagent solution that the present invention uses is mixed with following pair of agent reagent:

The reagent of forming by described damping fluid, stabilizing agent, coenzyme, acetyl phosphate, hydrogen peroxide and glyceraldehyde-3-phosphate 1;

The reagent of forming by described damping fluid, stabilizing agent, hydrogen cyanide synthase, pyruvate oxidase and glyceraldehyde-3-phosphate dehydrogenase 2;

Wherein coenzyme, hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase, acetyl phosphate, hydrogen peroxide, the position of glyceraldehyde-3-phosphate in reagent 1 or reagent 2 do not limit.

According to another kind preferred implementation of the present invention, the reagent that the present invention uses is mixed with following three doses of reagent:

The reagent of forming by described damping fluid, stabilizing agent, coenzyme, acetyl phosphate, hydrogen peroxide and glyceraldehyde-3-phosphate 1;

The reagent of forming by described damping fluid, stabilizing agent, pyruvate oxidase and glyceraldehyde-3-phosphate dehydrogenase 2;

The reagent of forming by described damping fluid, stabilizing agent and hydrogen cyanide synthase 3;

Wherein coenzyme, hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase, acetyl phosphate, hydrogen peroxide, the position of glyceraldehyde-3-phosphate in reagent 1, reagent 2 or reagent 3 do not limit.

The determining instrument that uses in glycocoll assay method of the present invention can be the ultraviolet analyser, for example 723 visible spectrophotometers of the ultraviolet-visible pectrophotometer of Shanghai precision instrumentation company limited sale, the sale of Tianjin Ka Nasi optic analytical instrument company limited; Semi-automatic biochemical analyzer, for example the BTS-330 semi-automatic biochemical analyzer of medical equipment company limited of Shanghai Victory-idea; Automatic clinical chemistry analyzer, for example: the automatic clinical chemistry analyzer of selling with trade name CX20 with trade name CB8000 or Beckman company with trade name 7600, Abbott with trade name 120, Hitachi, Ltd with trade name AU400, Toshiba with trade name BS-300, Olympus company by Mai Rui company.

The invention still further relates to glycocoll and measure kit.This glycocoll mensuration kit is made up of following powdered reagent, and water dissolves the liquid reagent that can directly use that obtains having following concentration range with their in use:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Coenzyme 0.1-5mmol/L

Hydrogen cyanide synthase 1000-80000U/L

Pyruvate oxidase 1000-80000U/L

Glyceraldehyde-3-phosphate dehydrogenase 1000-80000U/L

Acetyl phosphate 1-100mmol/L

Hydrogen peroxide 1-100mmol/L

Glyceraldehyde-3-phosphate 1-100mmol/L.

According to a kind of preferred implementation of the present invention, glycocoll of the present invention is measured kit to be had:

Reagent 1:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

DPN diphosphopyridine nucleotide .25mmol/L

Acetyl phosphate 5mmol/L

Hydrogen peroxide 15mmol/L

Glyceraldehyde-3-phosphate 5mmol/L;

Reagent 2 composed as follows:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Hydrogen cyanide synthase 16000U/L

Pyruvate oxidase 18000U/L

Glyceraldehyde-3-phosphate dehydrogenase 12000U/L.

According to another kind preferred implementation of the present invention, glycocoll of the present invention is measured kit to be had:

Reagent 1:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

DPN diphosphopyridine nucleotide .25mmol/L

Acetyl phosphate 5mmol/L

Hydrogen peroxide 15mmol/L

Glyceraldehyde-3-phosphate 5mmol/L;

Reagent 2 composed as follows:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Pyruvate oxidase 18000U/L

Glyceraldehyde-3-phosphate dehydrogenase 12000U/L;

Reagent 3 composed as follows:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Hydrogen cyanide synthase 16000U/L.

According to another kind preferred implementation of the present invention, measure in the kit at glycocoll of the present invention, described damping fluid is one or more damping fluids that are selected from three (ethyloic) aminomethane-hydrochloride buffer, phosphate buffer, imidazoles-hydrochloride buffer, sodium hydrogen phosphate-citrate buffer solution, borax-hydrochloride buffer, barbital sodium-hydrochloride buffer, boric acid-borate buffer solution, diethanolamine buffer or " PBS " damping fluid.

Preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphate buffer or " PBS " damping fluid.

More preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid or phosphate buffer.

In the present invention, described stabilizing agent is one or more stabilizing agents that are selected from ammonium sulfate, sodium chloride, ethylene glycol, propylene glycol, glycerine or sodium Diacetate or Sodium azide antiseptic.

Preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from ammonium sulfate, sodium chloride, propylene glycol, glycerine, sodium Diacetate or Sodium azide.

More preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from glycerine, ammonium sulfate or sodium Diacetate.

In the present invention, no matter be single agent reagent, two agent reagent or three doses of reagent, to measure in the method for glycocoll in the present invention, described coenzyme can be that one or more are selected from NADP ⁺, NAD ⁺Or thio-NAD ⁺Coenzyme.

When using glycocoll of the present invention to measure kit, can use the ultraviolet analyser, for example 723 visible spectrophotometers of the ultraviolet-visible pectrophotometer of Shanghai precision instrumentation company limited sale, the sale of Tianjin Ka Nasi optic analytical instrument company limited; Semi-automatic biochemical analyzer, for example the BTS-330 semi-automatic biochemical analyzer of medical equipment company limited of Shanghai Victory-idea; Automatic clinical chemistry analyzer, for example: the automatic clinical chemistry analyzer of selling with trade name CX20 with trade name CB8000 or Beckman company with trade name 7600, Abbott with trade name 120, Hitachi, Ltd with trade name AU400, Toshiba with trade name BS-300, Olympus company by Mai Rui company.

When adopting the inventive method to measure glycocoll, carry out test of many times according to requirement of experiment, these test findings that will obtain then calculate precision (CV) according to following formula:

$S=\sqrt{\mathrm{\Σ}{(\stackrel{\‾}{X}-X_i)}^2/n-1}$

In the formula:

-test findings mean value;

Xi-each time test findings;

N-test number (TN) .N 〉=10

$\mathrm{CV}=S/\stackrel{\‾}{X}*100\%$

These test findings that obtain are calculated relative extreme difference according to following formula:

$\mathrm{\Δ}=\frac{\stackrel{\‾}{U}-\stackrel{\‾}{V}}{(\stackrel{\‾}{X}+\stackrel{\‾}{Y}+\stackrel{\‾}{Z})\÷3}$

First each time test findings mean value

Second batch of each time test findings mean value

The 3rd batch of each time test findings mean value

Be X, Y, the maximal value among the Z

Be X, Y, the minimum value among the Z

Every Lot sample number is got n 〉=3

Determining through a large amount of tests, is the sample of 0-2mmol/L for glycocoll content, its analytical error can reach≤and 5%.

The sensitivity of the inventive method can reach 0.0001mmol/L.

Determine that by a large amount of tests it is 0-2mmol/L that the inventive method is measured the glycocoll content range, the inventive method is suitable for food inspection.

[embodiment]

Following embodiment illustrates the present invention and does not limit protection scope of the present invention.

Embodiment 1: glycocoll Determination on content in the milk

1) preparation of standard model

With a certain amount of glycocoll in the water-soluble or damping fluid, again glycine concentration is adjusted to 1 mM/liter;

2) testing sample pre-service

Milk sample is as testing sample, need not pre-service;

3) blank sample

Described water or damping fluid be as blank sample, its glycine concentration be 0 mM/liter;

The preparation of B, reagent solution:

It is single agent reagent that the glycocoll of present embodiment is measured reagent, and it contains:

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

Glycerine 1mol/L

NADP ⁺ 1.25mmol/L

Hydrogen cyanide synthase 16000U/L

Pyruvate oxidase 18000U/L

Glyceraldehyde-3-phosphate dehydrogenase 12000U/L

Acetyl phosphate 5mmol/L

Hydrogen peroxide 13mmol/L

Glyceraldehyde-3-phosphate 4mmol/L.

According to above-mentioned concentration these reagent being added the whole dissolvings of deionized water prepares stand-by.

C, milk sample to be measured, with at step B) reagent solution that obtains mixes according to volume ratio 1/50, in the reaction 5 minutes down of 37 ℃ of temperature, under predominant wavelength 340nm and commplementary wave length 405nm, measure, measure its absorbance over time Δ A (sample) be 0.0498;

D, with step C) determination step A under the same condition) and standard model absorbance over time Δ A (standard) be 0.1638;

E, with step C) be determined at steps A under the same condition) and the water that uses as the absorbance of blank solution over time Δ A (blank) be 0.0011;

F, data processing

By step C-E) absorbance of the predominant wavelength 340nm of described mensuration over time, calculate the content of glycocoll according to following formula:

In the formula:

Δ A (sample) expression step C) absorbance of the testing sample that obtains changes;

Δ A (blank) represents step e) absorbance of the blank sample that obtains changes;

Δ A (standard) expression step D) absorbance of standard model changes.

The glycocoll content that calculates this milk sample by following formula is 0.30mmol/L, and its error is ± 0.002mmol/L.

Embodiment 2: glycocoll Determination on content in the milk

1) preparation of standard model

A certain amount of glycocoll is soluble in water, again glycine concentration is adjusted to 1 mM/liter, as standard model;

2) preparation of testing sample

Milk sample need not be handled as testing sample;

3) blank sample

Described water is as blank sample, its glycine concentration be 0 mM/liter;

The preparation of B, reagent solution:

It is two agent reagent that glycocoll is measured reagent, and it contains:

Reagent 1 composed as follows:

Phosphate buffer 1 00mmol/L

NAD ⁺ 2.25mmol/L

Acetyl phosphate 5mmol/L

Hydrogen peroxide 15mmol/L

Glyceraldehyde-3-phosphate 5mmol/L;

Reagent 2 composed as follows:

Phosphate buffer 1 00mmol/L

Ethylene glycol 5mol/L

Hydrogen cyanide synthase 16000U/L

Pyruvate oxidase 32000U/L

Glyceraldehyde-3-phosphate dehydrogenase 40000U/L.

According to above-mentioned concentration these reagent being added the whole dissolvings of deionized water prepares stand-by.

C, sample use Hitachi's 7080 automatic clinical chemistry analyzers to measure

This automatic clinical chemistry analyzer main operating parameters is as follows:

Metering method: 2 end-point methods

Test and measuring time point: 21,31

Predominant wavelength: 340

Commplementary wave length: 405

Temperature of reaction: 37

Sample volume: 5

Reagent 1 (R1) volume: 200

Reagent 2 (R3) volume: 50

The Direction of Reaction: just

Standard model 1: 0.00

Standard model 2: 1.00

The calibration result shows that the K value is 611, and being converted into sensitivity is 0.1637 Δ A/mmol/L.

Test result shows that containing glycocoll in this milk is 0.53mmol/L.Its error is ± 0.003mmol/L.

Embodiment 3: glycocoll Determination on content in the milk sample

The mode of operation of this embodiment is identical with embodiment 2, just present embodiment:

The preparation of B, reagent solution:

It is three doses of reagent that glycocoll is measured reagent, and it contains:

Reagent 1 composed as follows:

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

NAD ⁺ 1.25mmol/L

Acetyl phosphate 6mmol/L

Hydrogen peroxide 15mmol/L

Glyceraldehyde-3-phosphate 4mmol/L;

Reagent 2 composed as follows:

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

Ammonium sulfate 1mol/L

Pyruvate oxidase 28000U/L

Glyceraldehyde-3-phosphate dehydrogenase 50000U/L;

Reagent 3 composed as follows:

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

Ammonium sulfate 2mol/L

Hydrogen cyanide synthase 26000U/L.

According to above-mentioned concentration these reagent being added the whole dissolvings of deionized water prepares stand-by.

C, sample use Hitachi's 7080 automatic clinical chemistry analyzers to measure

This automatic clinical chemistry analyzer main operating parameters is as follows:

Metering method: 2 end-point methods

Test and measuring time point: 21,31

Predominant wavelength: 340

Commplementary wave length: 405

Temperature of reaction: 37

Sample volume: 5

Reagent 1 (R1) volume: 20

Reagent 2 (R2) volume: 180

Reagent 3 (R3) volume: 50

The Direction of Reaction: just

Standard model 1: 0.00

Standard model 2: 1.00

The calibration result shows that the K value is 605, and being converted into sensitivity is 0.1653 Δ A/mmol/L.

Test result shows that containing glycocoll in this milk is 0.51mmol/L.Its error is ± 0.001mmol/L.

Embodiment 4: stability test

The mode of operation of this embodiment is identical with embodiment 1, and just after embodiment 1 implemented, the reagent that embodiment 1 uses had been deposited under 2-8 ℃ half a year and 1 year in airtight reagent bottle.

Use the same standard sample, use similarly to Example 2 novel agent and the above-mentioned reagent of depositing half a year and 1 year to measure respectively, other condition is all identical with embodiment 2, and its measurement result is as follows:

Table 1

?	Glycocoll content	Analytical error
			Novel agent	1.00mmol/L	±0.001mmol/L
Deposit the reagent of half a year	1.04mmol/L	±0.001mmol/L
			Deposit the reagent in 1 year	1.00mmol/L	±0.001mmol/L

Table 1 is the result show, uses kit of the present invention, adopts assay method of the present invention can guarantee to obtain stable result, and its stability is more than at least one year.

Embodiment 5: linear test

The mode of operation of this embodiment is identical with embodiment 2, just prepares new standard model concentration and reaches 2.50mmol/L, and the glycocoll of 2.50mmol/L according to doubling dilution, is tested then, and its measurement result is as follows:

Table 2

The glycocoll desired value	The glycocoll test value
		0.00	0.01
0.25	0.24
		0.50	0.51
0.75	0.75
		1.00	1.02
1.25	1.25
		1.50	1.49
2.00	2.01
		2.50	2.40

Table 2 is the result show, uses kit of the present invention, adopts assay method of the present invention can guarantee that linearity can reach 2.00mmol/L.

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)≤0.002; Absorbance time response curve should be the rising curve; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 2.00mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤5% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.1600 ± 0.0080A/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. glycine concentration assay method that utilizes two times of TRAP, enzymic colorimetric and enzyme-linked method technology, the know-why of its mensuration is to finish according to the serial catalytic reaction of following hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase:

Glycocoll+2 coenzyme Hydrogen cyanide synthaseHydrogen cyanide+carbon dioxide+2 reduced coenzymes

Carbon dioxide+acetyl phosphate+hydrogen peroxide Pyruvate oxidasePhosphate radical+pyruvic acid

+ oxygen+water

Phosphate radical+glyceraldehyde-3-phosphate+coenzyme Glyceraldehyde-3-phosphate dehydrogenase

Glyceric acid-1,3-bis phosphoric acid+reduced coenzyme.

2. the assay method of a glycocoll is characterized in that the step of this method is as follows:

2.1 sample is prepared:

2.1.1 the preparation of standard model

With a certain amount of glycocoll in the water-soluble or damping fluid, again its concentration is adjusted to 1 mM/liter;

2.1.2 the preparation of testing sample

The testing liquid sample is directly tested, need not pre-service; With a certain amount of solid sample to be measured as the preparation standard sample in the water-soluble or damping fluid;

2.1.3 blank sample

Described water or damping fluid be as blank sample, its glycine concentration be 0 mM/liter;

2.2 the preparation of reagent solution:

Pipette or take by weighing damping fluid, stabilizing agent, coenzyme, hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase, acetyl phosphate, hydrogen peroxide and glyceraldehyde-3-phosphate respectively, then they are mixed, the water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-5mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/L, 1-100mmol/L, 1-100mmol/L and 1-100mmol/L;

2.3 testing sample with in step 2.2) reagent solution that obtains mixes according to volume ratio 1/10 to 1/500, reacted 5-60 minute down at temperature 15-45 ℃, at predominant wavelength 340nm and commplementary wave length 405nm (if limited by instrument, can not establish commplementary wave length) under measure, measure its absorbance over time;

2.4 with step 2.3) determination step 2.1.1 under the same condition) and standard model absorbance over time;

2.5 with step 2.3) be determined at step 2.1.3 under the same condition) water that uses or damping fluid are as the absorbance of blank solution over time;

2.6 data processing

By step 2.3-2.5) absorbance of the predominant wavelength 340nm of described mensuration over time, calculate the content of glycocoll according to following formula:

In the formula:

Δ A (sample) represents step 2.3) absorbance of the testing sample that obtains changes;

Δ A (blank) represents step 2.5) absorbance of the blank solution that obtains changes;

Δ A (standard) represents step 2.4) absorbance of standard model changes.

3. according to claim 1,2 described assay methods, when it is characterized in that using automatic clinical chemistry analyzer to measure, testing sample, described standard model and described blank sample are by the sampling automatically under imposing a condition of this analyser, measure under the following conditions then: assay method is an end-point method, 37 ℃ of temperature, 10 minutes reaction time, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glycocoll sample and reagent is 1/10-1/500, the Direction of Reaction is positive reaction, 1/0 minute time delay, 4/5 minute detection time.

4. according to claim 1,2 or 3 described assay methods, it is characterized in that described stabilizing agent is one or more stabilizing agents that are selected from ammonium sulfate, sodium chloride, ethylene glycol, propylene glycol, glycerine or antiseptics such as sodium Diacetate, Sodium azide; Described damping fluid is one or more damping fluids that are selected from three (ethyloic) aminomethane-hydrochloride buffer, phosphate buffer, imidazoles-hydrochloride buffer, sodium hydrogen phosphate-citrate buffer solution, borax-hydrochloride buffer, barbital sodium-hydrochloride buffer, boric acid-borate buffer solution, diethanolamine buffer or " PBS " damping fluid; Described coenzyme is that one or more are selected from NADP ⁺, NAD ⁺Or thio-NAD ⁺Coenzyme.

5. according to claim 1,2 or 3 described assay methods, it is characterized in that:

5.1 described reagent solution is mixed with as the agent reagent that places an order:

It is made up of described damping fluid, stabilizing agent, coenzyme, acetyl phosphate, hydrogen peroxide, glyceraldehyde-3-phosphate, hydrogen cyanide synthase, pyruvate oxidase and glyceraldehyde-3-phosphate dehydrogenase;

5.2 described reagent solution is mixed with following pair of agent reagent:

Reagent 1, it is made up of described damping fluid, stabilizing agent, coenzyme, acetyl phosphate, hydrogen peroxide and glyceraldehyde-3-phosphate;

Reagent 2, it is made up of described damping fluid, stabilizing agent, hydrogen cyanide synthase, pyruvate oxidase and glyceraldehyde-3-phosphate dehydrogenase;

Wherein coenzyme, hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase, acetyl phosphate, hydrogen peroxide, the position of glyceraldehyde-3-phosphate in reagent 1 or reagent 2 do not limit.

5.3 described reagent is mixed with following three doses of reagent:

Reagent 1, it is made up of described damping fluid, stabilizing agent, coenzyme, acetyl phosphate, hydrogen peroxide and glyceraldehyde-3-phosphate;

Reagent 2, it is made up of described damping fluid, stabilizing agent, pyruvate oxidase and glyceraldehyde-3-phosphate dehydrogenase;

Reagent 3, it is made up of described damping fluid, stabilizing agent and hydrogen cyanide synthase;

Wherein coenzyme, hydrogen cyanide synthase, pyruvate oxidase, glyceraldehyde-3-phosphate dehydrogenase, acetyl phosphate, hydrogen peroxide, the position of glyceraldehyde-3-phosphate in reagent 1, reagent 2 or reagent 3 do not limit.

6. a glycocoll is measured kit, it is characterized in that:

6.1 it is made up of following powdered reagent, water dissolves the liquid reagent that can directly use that obtains having following concentration range with their in use:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Coenzyme 0.1-5mmol/L

Hydrogen cyanide synthase 1000-80000U/L

Pyruvate oxidase 1000-80000U/L

Glyceraldehyde-3-phosphate dehydrogenase 1000-80000U/L

Acetyl phosphate 1-100mmol/L

Hydrogen peroxide 1-100mmol/L

Glyceraldehyde-3-phosphate 1-100mmol/L.

6.2 measure kit according to the described glycocoll of claim 6.1, it is characterized in that it has:

Reagent 1 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Coenzyme 0.1-5mmol/L

Acetyl phosphate 1-100mmol/L

Hydrogen peroxide 1-100mmol/L

Glyceraldehyde-3-phosphate 1-100mmol/L;

Reagent 2 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Hydrogen cyanide synthase 1000-80000U/L

Pyruvate oxidase 1000-80000U/L

Glyceraldehyde-3-phosphate dehydrogenase 1000-80000U/L.

6.3 measure kit according to the described glycocoll of claim 6.1, it is characterized in that it has:

Reagent 1 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Coenzyme 0.1-5mmol/L

Acetyl phosphate 1-100mmol/L

Hydrogen peroxide 1-100mmol/L

Glyceraldehyde-3-phosphate 1-100mmol/L;

Reagent 2 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Pyruvate oxidase 1000-80000U/L

Glyceraldehyde-3-phosphate dehydrogenase 1000-80000U/L;

Reagent 3 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Hydrogen cyanide synthase 1000-80000U/L.