CN102087288A - Determination method of galactose and kit 9044 for diagnosing/determining galactose - Google Patents

Abstract

The invention relates to a determination method of galactose content by adopting an enzymatic colorimetric method and an enzyme-linked immuno sorbent assay technology, composition and components of a reagent, and a kit for diagnosing/determining the galactose. The determination method has the advantages of high sensitivity and small error. Therefore, the determination method and the kit disclosed by the invention can be widely applied to clinical medicine/food inspection.

Description

The assay method of galactose and galactose diagnosis/determination kit--9044

[technical field]

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

[background technology]

Milk have the laudatory title of " liquid golden " in nutrition circle, China united proposition as far back as 1992 by seven positions: the slogan of " one glass of strong nationality of milk ".Milk almost contains the various nutrients that needed by human body is wanted except that fibre-bearing element not.After lactose in milk enters human body, resolve into glucose and galactose through the effect of small intestine lactase.Galactose is the essential material of baby's brain development, with the substantial connection that shot up of baby's brain.

Though milk good but not all people can both enjoy, some people is because the shortage of lactase, human body can not well be digested and assimilated the nutrient in the milk, also can cause the malabsorption of elements such as calcium, phosphorus, potassium, iron, influence infant's physique and intelligence development, in the elderly, alactasia is to cause osteoporotic major reason.Chinese adult is drunk the incidence of disease of lactose malabsorption after the cow's milk up to 86.7%, and not tolerating index is 0.9.

For fear of the infringement that inappropriate drink milk causes health, check oneself whether to tolerate lactose before therefore being preferably in drink milk, behind milk drink, the concentration of galactose in the test urine, this is at present external the most frequently used a kind of method, and is more easy, economical, reliably.

[summary of the invention]

[technical matters that will solve]

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

Another object of the present invention provides a kind of galactose diagnosis/determination kit.

[technical scheme]

The present invention is achieved through the following technical solutions.

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 the mensuration galactose in the absorbance at 340nm wavelength place the method for measuring.

Galactose assay method of the present invention know-why be to finish for the serial catalytic reaction of biotin synzyme, malonic acid-semialdehyde dehydrogenasa according to following galactokinase, desulfuration:

Galactose+adenosine triphosphate GalactokinaseAdenosine diphosphate+galactose 1-phosphoric acid

Adenosine diphosphate+phosphate radical+desulfuration is for biotin Desulfuration is for the biotin synzyme

Adenosine triphosphate+7,8-diaminopelargonic acid+carbon dioxide

Carbon dioxide+acetyl-coacetylase+reduced coenzyme Malonic acid-semialdehyde dehydrogenasa

3-formyl acetic acid+coacetylase+coenzyme

Method of the present invention is utilized galactokinase (galactokinase; EC 2.7.1.6) enzyme (idol) connection desulfuration is for biotin synzyme (dethiobiotin synthase; EC 6.3.3.3), malonic acid-semialdehyde dehydrogenasa (malonate-semialdehyde dehydrogenase; EC 1.2.1.18) enzymatic reaction end-point method.The reaction of galactokinase enzymolysis galactose produces adenosine diphosphate, again by of the effect of (idol) associating desulfuration for biotin synzyme, malonic acid-semialdehyde dehydrogenasa, 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, can can calculate the concentration of galactose by measuring the degree that 340nm place absorbance descends like this.

The present invention is achieved through the following technical solutions.

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

A, sample are prepared:

A.1 the preparation of standard model

A certain amount of galactose in the water-soluble or damping fluid, is adjusted to galactose concentration 100 micromoles per liter again, and the solution that obtains is as standard model;

A.2 testing sample pre-service

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 galactose concentration is 0 micromoles per liter;

The preparation of B, reagent solution:

Pipette or take by weighing damping fluid respectively, stabilizing agent, reduced coenzyme, galactokinase, desulfuration is for the biotin synzyme, malonic acid-semialdehyde dehydrogenasa, adenosine triphosphate, unit price phosphate (phosphate radical), desulfuration is for biotin and acetyl-coacetylase, then they are mixed, the water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-0.35mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/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 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 galactose 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 galactose 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, 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 galactose 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 reduced coenzyme is one or more reduced coenzymes that are selected from NADPH, NADH or thio-NADH.

According to a kind of preferred implementation of the present invention, when the present invention uses automatic clinical chemistry analyzer to measure galactose, 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 galactose sample and reagent is 1/10-1/500, the Direction of Reaction is negative 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 for biotin and acetyl-coacetylase by described damping fluid, stabilizing agent, reduced coenzyme, adenosine triphosphate, unit price phosphate, desulfuration 1;

By described damping fluid, stabilizing agent, galactokinase, desulfuration for biotin synzyme and reagent 2 that malonic acid-the semialdehyde dehydrogenasa is formed;

Wherein reduced coenzyme, galactokinase, desulfuration do not limit for biotin, the position of acetyl-coacetylase in reagent 1 or reagent 2 for biotin synzyme, malonic acid-semialdehyde dehydrogenasa, adenosine triphosphate, unit price phosphate, desulfuration.

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 for biotin and acetyl-coacetylase by described damping fluid, stabilizing agent, reduced coenzyme, adenosine triphosphate, unit price phosphate, desulfuration 1;

By described damping fluid, stabilizing agent, desulfuration for biotin synzyme and reagent 2 that malonic acid-the semialdehyde dehydrogenasa is formed;

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

Wherein reduced coenzyme, galactokinase, desulfuration do not limit for biotin, the position of acetyl-coacetylase in reagent 1, reagent 2 or reagent 3 for biotin synzyme, malonic acid-semialdehyde dehydrogenasa, adenosine triphosphate, unit price phosphate, desulfuration.

The determining instrument that uses in galactose 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 the galactose diagnosis/determination kit.This galactose 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

Reduced coenzyme 0.1-0.35mmol/L

Galactokinase 1000-80000U/L

Desulfuration is for biotin synthetase 1 000-80000U/L

Malonic acid-semialdehyde dehydrogenase 10 00-80000U/L

Adenosine triphosphate 1-100mmol/L

Unit price phosphate 1-100mmol/L

Desulfuration is for biotin 1-100mmol/L

Acetyl-coacetylase 1-100mmol/L.

According to a kind of preferred implementation of the present invention, galactose diagnosis/determination kit of the present invention has:

Reagent 1:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Adenosine triphosphate 3mmol/L

Unit price phosphate 15mmol/L

Desulfuration is for biotin 5mmol/L

Acetyl-coacetylase 15mmol/L;

Reagent 2 composed as follows:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Galactokinase 16000U/L

Desulfuration is for biotin synthetase 1 8000U/L

Malonic acid-semialdehyde dehydrogenase 1 2000U/L.

According to another kind preferred implementation of the present invention, galactose diagnosis/determination kit of the present invention has:

Reagent 1:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Adenosine triphosphate 3mmol/L

Unit price phosphate 15mmol/L

Desulfuration is for biotin 5mmol/L

Acetyl-coacetylase 15mmol/L;

Reagent 2 composed as follows:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Desulfuration is for biotin synthetase 1 8000U/L

Malonic acid-semialdehyde dehydrogenase 1 20000U/L;

Reagent 3 composed as follows:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Galactokinase 16000U/L.

According to another kind preferred implementation of the present invention, in galactose diagnosis/determination kit 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, 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 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 galactose in the present invention, described reduced coenzyme can be one or more reduced coenzymes that are selected from NADPH, NADH or thio-NADH.

When using galactose diagnosis/determination kit of the present invention, 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 L20, 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 galactose, 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:

X-test findings mean value;

Each time of Xi-test findings;

N-test number (TN) .N 〉=10

CV＝S/X*100％

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

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

X---the-batch each time test findings mean value

Y---second batch of each time test findings mean value

Z---the 3rd batch of each time test findings mean value

U---be X, Y, the maximal value among the Z

V---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 0-600 μ mol/L for galactose content, its analytical error can reach≤and 6%.

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

Determine that by a large amount of tests it is 0-600 μ mol/L that the inventive method is measured the galactose content scope, the inventive method is suitable for clinical medicine/food diagnosis/detection.

[embodiment]

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

Embodiment 1: the mensuration of galactose content in the blood plasma

1) preparation of standard model

A certain amount of galactose in the water-soluble or damping fluid, is adjusted to galactose concentration 100 micromoles per liter 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 galactose concentration is 0 micromoles per liter;

The preparation of B, reagent solution:

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

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

Glycerine 1mol/L

NADPH 0.25mmol/L

Galactokinase 16000U/L

Desulfuration is for biotin synthetase 1 8000U/L

Malonic acid-semialdehyde dehydrogenase 1 2000U/L

Adenosine triphosphate 5mmol/L

Unit price phosphate 15mmol/L

Desulfuration is for biotin 3mmol/L

Acetyl-coacetylase 14mmol/L.

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

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 0.0097;

D, with step C) determination step A under the same condition) and standard model absorbance over time Δ A (standard) be 0.0155;

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 0.0006;

F, data processing

By step C-E) absorbance of the predominant wavelength 340nm of described mensuration over time, calculate the content of galactose 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 galactose content that calculates this plasma sample by following formula is 61 μ mol/L, and its error is ± 4 μ mol/L.

Embodiment 2: the mensuration of galactose content in the blood plasma

1) preparation of standard model

A certain amount of galactose is soluble in water, again galactose 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 galactose concentration is 0 micromoles per liter;

The preparation of B, reagent solution:

Galactose diagnosing/determining reagent is two agent reagent, and it contains:

Reagent 1 composed as follows:

Phosphate buffer 1 00mmol/L

NADH 0.25mmol/L

Adenosine triphosphate 5mmol/L

Desulfuration is for biotin 3mmol/L

Acetyl-coacetylase 14mmol/L;

Reagent 2 composed as follows:

Phosphate buffer 1 00mmol/L

Ethylene glycol 5mol/L

Galactokinase 16000U/L

Desulfuration is for biotin synzyme 32000U/L

Malonic acid-semialdehyde dehydrogenasa 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: 20

Reagent 1 (R1) volume: 200

Reagent 2 (R3) volume: 50

The Direction of Reaction: negative

Standard model 1: 0

Standard model 2: 100

The calibration result shows that the K value is-4985, and being converted into sensitivity is 0.00020 Δ A/ μ mol/L.

Test result shows that containing galactose in this blood plasma is 29. μ mol/L.Its error is ± 1 μ mol/L.

Embodiment 3: the mensuration of galactose content in the plasma sample

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

The preparation of B, reagent solution:

Galactose diagnosing/determining reagent is three doses of reagent, and it contains:

Reagent 1 composed as follows:

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

NADH 0.25mmol/L

Adenosine triphosphate 5mmol/L

Unit price phosphate 15mmol/L

Desulfuration is for biotin 3mmol/L

Acetyl-coacetylase 14mmol/L;

Reagent 2 composed as follows:

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

Ammonium sulfate 1mol/L

Desulfuration is for biotin synzyme 28000U/L

Malonic acid-semialdehyde dehydrogenasa 50000U/L;

Reagent 3 composed as follows:

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

Ammonium sulfate 2mol/L

Galactokinase 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: 20

Reagent 1 (R1) volume: 20

Reagent 2 (R2) volume: 180

Reagent 3 (R3) volume: 50

The Direction of Reaction: negative

Standard model 1: 0

Standard model 2: 100

The calibration result shows that the K value is-4805, and being converted into sensitivity is 0.00021 Δ A/ μ mol/L.

Test result shows that containing galactose in this blood plasma is 73 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 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

	Galactose 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

200μmol/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 800 μ mol/L, and the galactose of 800 μ mol/L according to doubling dilution, is tested then, and its measurement result is as follows:

Table 2

The galactose desired value	The galactose test value
		0	0
100	99
		200	200

300	298
		400	399
500	501
		600	598
700	675
		800	718

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 600 μ mol/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)≤0.01; Absorbance time response curve should be decline curve; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 600 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, the linear range broadness, and stationary phase is long, is enough to easy to utilize.

Claims (6)

1. galactose concentration assay method that utilizes enzymic colorimetric and enzyme-linked method technology, the know-why of its mensuration is to finish for the serial catalytic reaction of biotin synzyme, malonic acid-semialdehyde dehydrogenasa according to following galactokinase, desulfuration:

Galactose+adenosine triphosphate GalactokinaseAdenosine diphosphate+galactose 1-phosphoric acid

Adenosine diphosphate+phosphate radical+desulfuration is for biotin Desulfuration is for the biotin synzyme

Adenosine triphosphate+7,8-diaminopelargonic acid+carbon dioxide

Carbon dioxide+acetyl-coacetylase+reduced coenzyme Malonic acid-semialdehyde dehydrogenasa

3-formyl acetic acid+coacetylase+coenzyme

2. the assay method of a galactose 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

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

2.1.2 testing sample pre-service

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 galactose concentration is 0 micromoles per liter;

2.2 the preparation of reagent solution:

Pipette or take by weighing damping fluid respectively, stabilizing agent, reduced coenzyme, galactokinase, desulfuration is for the biotin synzyme, malonic acid-semialdehyde dehydrogenasa, adenosine triphosphate, unit price phosphate (phosphate radical), desulfuration is for biotin and acetyl-coacetylase, then they are mixed, the water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-0.35mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/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 described mensuration over time, calculate the content of galactose 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 galactose sample and reagent is 1/10-1/500, the Direction of Reaction is negative 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, glycocoll-sodium hydrate buffer solution, barbital sodium-hydrochloride buffer, boric acid-borate buffer solution, diethanolamine buffer or " PBS " damping fluid; Described reduced coenzyme is one or more reduced coenzymes that are selected from NADPH, NADH or thio-NADH.

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 for biotin synzyme and malonic acid-semialdehyde dehydrogenasa for biotin, acetyl-coacetylase, galactokinase, desulfuration described damping fluid, stabilizing agent, reduced coenzyme, adenosine triphosphate, unit price phosphate, desulfuration;

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

Reagent 1, it is made up of for biotin and acetyl-coacetylase described damping fluid, stabilizing agent, reduced coenzyme, adenosine triphosphate, unit price phosphate, desulfuration;

Reagent 2, it is made up of for biotin synzyme and malonic acid-semialdehyde dehydrogenasa described damping fluid, stabilizing agent, galactokinase, desulfuration;

Wherein reduced coenzyme, galactokinase, desulfuration do not limit for biotin, the position of acetyl-coacetylase in reagent 1 or reagent 2 for biotin synzyme, malonic acid-semialdehyde dehydrogenasa, adenosine triphosphate, unit price phosphate, desulfuration.

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

Reagent 1, it is made up of for biotin and acetyl-coacetylase described damping fluid, stabilizing agent, reduced coenzyme, adenosine triphosphate, unit price phosphate, desulfuration;

Reagent 2, it is made up of for biotin synzyme and malonic acid-semialdehyde dehydrogenasa described damping fluid, stabilizing agent, desulfuration;

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

Wherein reduced coenzyme, galactokinase, desulfuration do not limit for biotin, the position of acetyl-coacetylase in reagent 1, reagent 2 or reagent 3 for biotin synzyme, malonic acid-semialdehyde dehydrogenasa, adenosine triphosphate, unit price phosphate, desulfuration.

6. galactose 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

Reduced coenzyme 0.1-0.35mmol/L

Galactokinase 1000-80000U/L

Desulfuration is for biotin synthetase 1 000-80000U/L

Malonic acid-semialdehyde dehydrogenase 10 00-80000U/L

Adenosine triphosphate 1-100mmol/L

Unit price phosphate 1-100mmol/L

Desulfuration is for biotin 1-100mmol/L

Acetyl-coacetylase 1-100mmol/L.

6.2, it is characterized in that it has according to the described galactose diagnosis/determination kit of claim 6.1:

Reagent 1 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Reduced coenzyme 0.1-0.35mmol/L

Adenosine triphosphate 1-100mmol/L

Unit price phosphate 1-100mmol/L

Desulfuration is for biotin 1-100mmol/L

Acetyl-coacetylase 1-100mmol/L;

Reagent 2 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Galactokinase 1000-80000U/L

Desulfuration is for biotin synthetase 1 000-80000U/L

Malonic acid-semialdehyde dehydrogenase 10 00-80000U/L.

6.3, it is characterized in that it has according to the described galactose diagnosis/determination kit of claim 6.1:

Reagent 1 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Reduced coenzyme 0.1-0.35mmol/L

Adenosine triphosphate 1-100mmol/L

Unit price phosphate 1-100mmol/L

Desulfuration is for biotin 1-100mmol/L

Acetyl-coacetylase 1-100mmol/L;

Reagent 2 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Desulfuration is for biotin synthetase 1 000-80000U/L

Malonic acid-semialdehyde dehydrogenase 10 00-80000U/L;

Reagent 3 composed as follows:

Damping fluid 20-500mmol/L

Stabilizing agent 0.001-7mol/L

Galactokinase 1000-80000U/L.