CN102539695A - Determination method of ammonia (ammonium ion) and diagnosis/determination reagent kit of ammonia (ammonium ion) - Google Patents

Abstract

The invention relates to a determination method for determining ammonia (ammonium ion) content by utilizing an enzymatic colorimetric method and enzyme couple reaction technology, and composition and components of a reagent. The technology principle for the determination is that the determination is finished according to series catalytic reactions of ammonia kinase, propionyl-CoA carboxylase and chlorobenzoic acid-1,2-dioxygenase. The invention further relates to a diagnosis/determination reagent kit of ammonia (an ammonium ion). The determination method is high in sensitivity and small in errors, and accordingly the determination method and the reagent kit can be widely applied to clinical medicine/food inspection.

Description

The assay method of ammonia (ammonium ion) and ammonia (ammonium ion) diagnosis/determination kit

Technical field

The present invention relates to medical science/Food Inspection determination techniques field, more specifically, the present invention relates to ammonia (ammonium ion) diagnosing/determining method and kit thereof.

Background technology

The ammonia method for measuring has microdiffusion, ion exchange process, enzyme process and ammonia electrode method etc.Use at present maximum methods and be enzyme process and based on the determination of blood ammonia appearance analytic approach of ion-selective electrode.

Diffusion method discharges NH after being the sample alkalization ₃, the ammonia that discharges with acidometric titration, or form the two mercury amine of pale brown look iodate with the Nessler reaction and carry out colorimetric.These methods need alkalization, and endogenic ammonia forms and impacts, and its accuracy and precision are affected, and seldom use at present; Ion exchange process is more accurate than diffusion method, and CV is 8%～13%; Ion selective electrode method is to utilize NH ₃Be diffused into electrode surface, the pH that causes electrode changes and measures, and the CV of this method is 3.5%～4.8%, and the recovery is high.In conjunction with concrete actual, should be practical with the enzymatic assays.

The retrieval Chinese patent is only found 87105593.7 patented claims and is disclosed a kind of rapid freezing cup for blood ammonia determination, does not but find more satisfactory determination of blood ammonia method.

Summary of the invention

[technical matters that will solve]

The assay method that the purpose of this invention is to provide a kind of ammonia (ammonium ion).

Another object of the present invention provides a kind of ammonia (ammonium ion) diagnosis/determination kit.

[technical scheme]

The present invention realizes through following technical proposals.

Method of the present invention is the coupling technique of a kind of employing enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction), utilizes reduced form nicotinamide coenzyme (reduced coenzyme) changes mensuration ammonia (ammonium ion) in the absorbance of 340nm wavelength the method for measuring.

Ammonia of the present invention (ammonium ion) assay method know-why be according to following ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1, the serial catalytic reaction of 2-dioxygenase is accomplished:

Ammonia+adenosine triphosphate Ammonia kinaseAdenosine diphosphate+phosphamide

Adenosine diphosphate+phosphate radical+methyl apple acyl-coacetylase Propionyl CoA carboxylase

Adenosine triphosphate+propionyl-coacetylase+carbon dioxide

Carbon dioxide+catechol+coenzyme+chlorine Chlorobenzoic acid-1, the 2-dioxygenase

2-chlorobenzoic acid+reduced coenzyme+oxygen

Method of the present invention is utilized ammonia kinase (ammonia kinase; EC 2.7.3.8) enzyme (idol) joins propionyl CoA carboxylase (propionyl-CoA carboxylase; EC 6.4.1.3), chlorobenzoic acid-1, (Chlorobenzoate 1,2-dioxygenase for the 2-dioxygenase; EC 1.14.12.13) enzymatic reaction colorimetric end-point method.Ammonia kinase enzymolysis ammonia react produces adenosine diphosphate; Again through (idol) associating propionyl CoA carboxylase, chlorobenzoic acid-1; The effect of 2-dioxygenase, coenzyme (not having absorption peak at the 340nm place) reduction becomes reduced coenzyme (absorption peak being arranged at the 340nm place) the most at last, thereby is able to measure the reduced coenzyme degree that absorbance rises at the 340nm place; Can calculate the concentration of ammonia (ammonium ion) through measuring the degree that 340nm place absorbance rises like this.

The present invention realizes through following technical proposals.

The present invention relates to the assay method of a kind of ammonia (ammonium ion).The step of this ammonia (ammonium ion) assay method is following:

A, sample are prepared:

A.1 the preparation of standard model

A certain amount of ammonia salt in the water-soluble or damping fluid, is adjusted to 100 micromoles per liter with ammonia concentration again, and 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 are as blank sample, and its ammonia (ammonium ion) concentration is 0 micromoles per liter;

The preparation of B, reagent solution:

Pipette or take by weighing damping fluid, stabilizing agent, coenzyme, ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1 respectively; 2-dioxygenase, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine and unit price phosphate; Then they are mixed; The water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-2mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/L, 1-100mmol/L, 1-100mmol/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 receive 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 said mensuration over time, obtain the content of ammonia according to computes:

In the formula:

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

Δ A (blank) representes 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 ammonia (ammonium ion) assay method, described damping fluid should be appreciated that it is to make it measure the solution of the pH kept stable (generally being 6.0-9.0) of medium.If this pH be higher than 9.0 or pH be lower than 6.0, 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, glycocoll-sodium hydrate buffer solution, 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 ammonia (ammonium ion) 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, makes 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 sodium chloride, monoethylene 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 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 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 used automatic clinical chemistry analyzer to measure ammonia (ammonium ion), testing sample, said standard model and said blank sample were by (parameter) sampling automatically under imposing a condition of this analyser; Under following condition, measure 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 ammonia (ammonium ion) 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 two agent reagent:

The reagent of forming by described damping fluid, stabilizing agent, coenzyme, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine and unit price phosphate 1;

By described damping fluid, stabilizing agent, ammonia kinase, propionyl CoA carboxylase and chlorobenzoic acid-1, the reagent 2 that the 2-dioxygenase is formed;

Wherein coenzyme, ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1,2-dioxygenase, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine, the position of unit price 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, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine and unit price phosphate 1;

By described damping fluid, stabilizing agent, propionyl CoA carboxylase and chlorobenzoic acid-1, the reagent 2 that the 2-dioxygenase is formed;

The reagent of forming by described damping fluid, stabilizing agent and ammonia kinase 3;

Wherein coenzyme, ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1,2-dioxygenase, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine, the position of unit price phosphate in reagent 1, reagent 2 or reagent 3 do not limit.

The determining instrument that in ammonia of the present invention (ammonium ion) assay method, uses 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 ammonia (ammonium ion) diagnosis/determination kit.This ammonia (ammonium ion) diagnosis/determination 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 0.1-2mmol/L

Ammonia kinase 1000-80000U/L

Propionyl CoA carboxylase 1000-80000U/L

Chlorobenzoic acid-1,2-dioxygenase 1000-80000U/L

Adenosine triphosphate 1-100mmol/L

Methyl apple acyl coenzyme A 1-100mmol/L

Catechol 1-100mmol/L

Chlorine 1-100mmol/L

Unit price phosphate 1-100mmol/L.

According to a kind of preferred implementation of the present invention, ammonia of the present invention (ammonium ion) diagnosis/determination kit has:

Reagent 1:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Coenzyme 0.25mmol/L

Adenosine triphosphate 5mmol/L

Methyl apple acyl coenzyme A 5mmol/L

Catechol 5mmol/L

Chlorine 5mmol/L

Unit price phosphate 5mmol/L;

Form following reagent 2:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Ammonia kinase 16000U/L

Propionyl CoA carboxylase 18000U/L

Chlorobenzoic acid-1,2-dioxygenase 12000U/L.

According to another kind preferred implementation of the present invention, ammonia of the present invention (ammonium ion) diagnosis/determination kit has:

Reagent 1:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Coenzyme 0.25mmol/L

Adenosine triphosphate 5mmol/L

Methyl apple acyl coenzyme A 5mmol/L

Catechol 5mmol/L

Chlorine 5mmol/L

Unit price phosphate 5mmol/L;

Form following reagent 2:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Propionyl CoA carboxylase 18000U/L

Chlorobenzoic acid-1,2-dioxygenase 12000U/L;

Form following reagent 3:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Ammonia kinase 16000U/L.

According to another kind preferred implementation of the present invention; In ammonia of the present invention (ammonium ion) diagnosis/determination kit, 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, glycocoll-sodium hydrate buffer solution, 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 sodium chloride, monoethylene 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 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 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 ammonia (ammonium ion) in the present invention, described coenzyme can be that one or more are selected from NADP ⁺, NAD ⁺Or thio-NAD ⁺Coenzyme.

When using ammonia of the present invention (ammonium ion) diagnosis/determination 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 ammonia (ammonium ion), carry out test of many times according to requirement of experiment, these test findings that will obtain then go out precision (CV) according to computes:

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

In the formula:

-test findings mean value;

Each time of Xi-test findings;

N-test number (TN) .N >=10

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

These test findings that obtain are gone out relative extreme difference according to computes:

-first each time test findings mean value

-second batch of each time test findings mean value

the-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

Confirming through a large amount of tests, is the sample of 0-1000 μ mol/L for ammonia (ammonium ion) content, its analytical error can reach≤and 5%.

The sensitivity of the inventive method can reach 1 μ mol/L.

Confirm that through a large amount of tests it is 0-1000 μ mol/L that the inventive method is measured ammonia (ammonium ion) content range, the inventive method is suitable for clinical medicine/food diagnosing.

Embodiment

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

Embodiment 1: ammonia in the blood plasma (ammonium ion) Determination on content

1) preparation of standard model

A certain amount of ammonia salt in the water-soluble or damping fluid, is adjusted to 100 micromoles per liter with ammonia concentration again;

2) testing sample pre-service

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

3) blank sample

Described water or damping fluid are as blank sample, and its ammonia concentration is 0 micromoles per liter;

The preparation of B, reagent solution:

The ammonia diagnosing/determining reagent of present embodiment is single agent reagent, and it contains:

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

Glycerine 1mol/L

NADP ⁺ 0.25mmol/L

Ammonia kinase 16000U/L

Propionyl CoA carboxylase 18000U/L

Chlorobenzoic acid-1,2-dioxygenase 12000U/L

Adenosine triphosphate 5mmol/L

Methyl apple acyl coenzyme A 5mmol/L

Catechol 5mmol/L

Chlorine 5mmol/L

Unit price phosphate 5mmol/L.

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

C, plasma sample to be measured; With at step B) reagent solution that obtains mixes according to volume ratio 1/20; 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 0.0277;

D, with step C) determination step A under the same condition) and standard model absorbance over time Δ A (standard) be 0.0523;

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 0.0106;

F, data processing

By step C-E) absorbance of the predominant wavelength 340nm of said mensuration over time, obtain the content of ammonia (ammonium ion) according to computes:

In the formula:

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

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

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

Ammonia (ammonium ion) content that calculates this plasma sample through following formula is 82 μ mol/L, and its error is ± 2 μ mol/L.

Embodiment 2: ammonia in the blood plasma (ammonium ion) Determination on content

1) preparation of standard model

A certain amount of ammonia salt is soluble in water, again ammonia (ammonium ion) concentration is adjusted to 100 micromoles per liter, as standard model;

2) preparation of testing sample

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

3) blank sample

Described water is as blank sample, and its ammonia (ammonium ion) concentration is 0 micromoles per liter;

The preparation of B, reagent solution:

Ammonia (ammonium ion) diagnosing/determining reagent is two agent reagent, and it contains:

Form following reagent 1:

Phosphate buffer 1 00mmol/L

NAD ⁺ 0.25mmol/L

Adenosine triphosphate 5mmol/L

Methyl apple acyl coenzyme A 5mmol/L

Catechol 5mmol/L

Chlorine 5mmol/L

Unit price phosphate 5mmol/L;

Form following reagent 2:

Phosphate buffer 1 00mmol/L

Monoethylene glycol 5mol/L

Ammonia kinase 16000U/L

Propionyl CoA carboxylase 32000U/L

Chlorobenzoic acid-1,2-dioxygenase 40000U/L.

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

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

This automatic clinical chemistry analyzer main operating parameters is following:

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: 20

Reagent 1 (R1) volume: 200

Reagent 2 (R3) volume: 50

The Direction of Reaction: just

Standard model 1: 0

Standard model 2: 100

The calibration result shows that the K value is 5011, and being converted into sensitivity is 0.00020 Δ A/ μ mol/L.

Test result shows that containing ammonia (ammonium ion) in this blood plasma is 21 μ mol/L.Its error is ± 1 μ mol/L.

Embodiment 3: ammonia in the plasma sample (ammonium ion) Determination on content

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

The preparation of B, reagent solution:

Ammonia (ammonium ion) diagnosing/determining reagent is three doses of reagent, and it contains:

Form following reagent 1:

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

NAD ⁺ 0.25mmol/L

Adenosine triphosphate 5mmol/L

Methyl apple acyl coenzyme A 5mmol/L

Catechol 5mmol/L

Chlorine 5mmol/L

Unit price phosphate 5mmol/L;

Form following reagent 2:

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

Sodium chloride 2mol/L

Propionyl CoA carboxylase 28000U/L

Chlorobenzoic acid-1,2-dioxygenase 50000U/L;

Form following reagent 3:

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

Sodium chloride 2mol/L

Ammonia kinase 26000U/L.

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

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

This automatic clinical chemistry analyzer main operating parameters is following:

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: 20

Reagent 1 (R1) volume: 20

Reagent 2 (R2) volume: 180

Reagent 3 (R3) volume: 50

The Direction of Reaction: just

Standard model 1: 0

Standard model 2: 100

The calibration result shows that the K value is 4705, and being converted into sensitivity is 0.00021 Δ A/ μ mol/L.

Test result shows that containing ammonia (ammonium ion) in this blood plasma is 78 μ mol/L.Its error is ± 2 μ mol/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 with embodiment 2 same novel agent and the above-mentioned reagent of depositing half a year and 1 year and measure respectively, other condition is all identical with embodiment 2, and it is following that it measures the result:

Table 1

?	Ammonia (ammonium ion) content	Analytical error
			Novel agent	201μmol/L	±4μmol/L
Deposit the reagent of half a year	202μmol/L	±4μmol/L
			Deposit the reagent in 1 year	205μmol/L	±4μmol/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 1200 μ mol/L, and the ammonia (ammonium ion) of 800 μ mol/L according to doubling dilution, is tested then, and it is following that it measures the result:

Table 2

Ammonia (ammonium ion) desired value	Ammonia (ammonium ion) test value
		0	0
50	49
		100	99
200	98
		400	399
600	601
		800	798
1000	1003
		1200	1150

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 1000 μ mol/L.

The applicant adopts other assay methods of putting down in writing in the above summary of the invention all can reach the object of the 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 proof: adopt assay method of the present invention can draw required mensuration result through general biochemical analyzer fully---the blank reagent absorbance changes (Δ A)≤0.01; Absorbance time response curve should be the rising curve; Reagent can be surveyed effectively, and (R>=0.99) linear range can reach 1000 μ mol/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 0.0002 ± 0.0001A/ μ mol/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, and linear range is broad, and stationary phase is long, is enough to easy to utilize.

Claims (6)

1. one kind is utilized enzymic colorimetric and technological ammonia (ammonium ion) method for measurement of concentration of enzyme-linked method, and the know-why of its mensuration is according to following ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1, and the serial catalytic reaction of 2-dioxygenase is accomplished:

Ammonia+adenosine triphosphate Ammonia kinaseAdenosine diphosphate+phosphamide

Adenosine diphosphate+phosphate radical+methyl apple acyl-coacetylase Propionyl CoA carboxylase

Adenosine triphosphate+propionyl-coacetylase+carbon dioxide

Carbon dioxide+catechol+coenzyme+chlorine Chlorobenzoic acid-1, the 2-dioxygenase

2-chlorobenzoic acid+reduced coenzyme+oxygen

2. the assay method of an ammonia (ammonium ion) is characterized in that the step of this method is following:

2.1 sample is prepared:

2.1.1 the preparation of standard model

A certain amount of ammonia salt in the water-soluble or damping fluid, is adjusted to 100 micromoles per liter with its concentration again;

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 are as blank sample, and its ammonia (ammonium ion) concentration is 0 micromoles per liter;

2.2 the preparation of reagent solution:

Pipette or take by weighing damping fluid, stabilizing agent, coenzyme, ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1 respectively; 2-dioxygenase, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine and unit price phosphate; Then they are mixed; The water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-2mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/L, 1-100mmol/L, 1-100mmol/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 said mensuration over time, obtain the content of ammonia according to computes:

In the formula:

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

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

Δ A (standard) representes 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, said standard model and said blank sample are by the sampling automatically under imposing a condition of this analyser; Under following condition, measure 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 ammonia 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 sodium chloride, monoethylene 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, glycocoll-sodium hydrate buffer solution, 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 like the agent reagent that places an order:

It is by described damping fluid, stabilizing agent, coenzyme, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine, unit price phosphate, ammonia kinase, propionyl CoA carboxylase and chlorobenzoic acid-1, and the 2-dioxygenase is formed;

5.2 described reagent solution is mixed with following two agent reagent:

Reagent 1, it is made up of described damping fluid, stabilizing agent, coenzyme, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine and unit price phosphate;

Reagent 2, it is by described damping fluid, stabilizing agent, ammonia kinase, propionyl CoA carboxylase and chlorobenzoic acid-1, the 2-dioxygenase is formed;

Wherein coenzyme, ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1,2-dioxygenase, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine, the position of unit price 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, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine and unit price phosphate;

Reagent 2, it is by described damping fluid, stabilizing agent, propionyl CoA carboxylase and chlorobenzoic acid-1, the 2-dioxygenase is formed;

Reagent 3, it is made up of described damping fluid, stabilizing agent and ammonia kinase;

Wherein coenzyme, ammonia kinase, propionyl CoA carboxylase, chlorobenzoic acid-1,2-dioxygenase, adenosine triphosphate, methyl apple acyl coenzyme A, catechol, chlorine, the position of unit price phosphate in reagent 1, reagent 2 or reagent 3 do not limit.

6. an ammonia (ammonium ion) diagnosis/determination kit 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 0.1-2mmol/L

Ammonia kinase 1000-80000U/L

Propionyl CoA carboxylase 1000-80000U/L

Chlorobenzoic acid-1,2-dioxygenase 1000-80000U/L

Adenosine triphosphate 1-100mmol/L

Methyl apple acyl coenzyme A 1-100mmol/L

Catechol 1-100mmol/L

Chlorine 1-100mmol/L

Unit price phosphate 1-100mmol/L;

6.2, it is characterized in that it has according to the described ammonia of claim 6.1 (ammonium ion) diagnosis/determination kit:

Form following reagent 1:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Coenzyme 0.1-2mmol/L

Adenosine triphosphate 1-100mmol/L

Methyl apple acyl coenzyme A 1-100mmol/L

Catechol 1-100mmol/L

Chlorine 1-100mmol/L

Unit price phosphate 1-100mmol/L;

Form following reagent 2:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Ammonia kinase 1000-80000U/L

Propionyl CoA carboxylase 1000-80000U/L

Chlorobenzoic acid-1,2-dioxygenase 1000-80000U/L;

6.3, it is characterized in that it has according to the described ammonia of claim 6.1 (ammonium ion) diagnosis/determination kit:

Form following reagent 1:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Coenzyme 0.1-2mmol/L

Adenosine triphosphate 1-100mmol/L

Methyl apple acyl coenzyme A 1-100mmol/L

Catechol 1-100mmol/L

Chlorine 1-100mmol/L

Unit price phosphate 1-100mmol/L;

Form following reagent 2:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Propionyl CoA carboxylase 1000-80000U/L

Chlorobenzoic acid-1,2-dioxygenase 1000-80000U/L;

Form following reagent 3:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Ammonia kinase 1000-80000U/L.