CN1746317A - Determination of adenosine deaminase activity and diagnostic kit of adenosine deaminase - Google Patents

Abstract

The invention is to determine the activity of the Adenosine deaminase and the reagent box of it. The box includes buffer, adenosine, phosphate, nucleoside phosphorylase, xanthine oxidase, peroxidase, urease, reduced chromogen and stabilizer. According to the proper ratio to mix the reagents, so the reagent can react and we can get the activity of the enzyme by detecting the absorbance of the main wave.

Description

Adenosine deaminase activity determination method and adenosine deaminase diagnosis reagent kit

Technical field

The present invention relates to a kind of method of measuring activity of adenosine deaminase, the invention still further relates to simultaneously, belong to medical test determination techniques field in order to realize the adenosine deaminase diagnosis reagent kit of this method.

Background technology

Medical research shows, adenosine deaminase is the nucleic acid metabolism enzyme that a kind of and body cell immunocompetence have important relationship.Therefore, the mensuration of activity of adenosine deaminase is used as good pernicious ascites pleural fluid, cerebrospinal fluid differential diagnosis, severe combined immunodeficiency disease (SCID), acute and chronic hepatitis, liver cirrhosis, the important diagnostic sign that diseases such as liver cancer are differentiated.

The activity determination method of adenosine deaminase mainly contains active nucleus method, physical method and biochemical process.Understand according to the applicant, generally adopt the UV-light method at present in the world, its measuring principle is: adenosine (white crystalline powder)+water (H2O) Adenosine deaminaseInosine (Inosine)+ammonium ion (NH4+), the inosine that this reflection process generates can be that the 265nm place manifests at wavelength, therefore can pass through the size of the big or small directly reflection activity of adenosine deaminase of this wavelength place absorbancy of mensuration.Yet this method can't be measured with the visible light analysis instrument of general hospital, and needs to use special ultraviolet light analyzer, therefore is difficult to apply conscientiously.

Retrieval finds, application number is 03128092.7, the applying date is that the Chinese patent application that 2003.05.29, name are called " a kind of reagent and method for making thereof of measuring adenosine deaminase " discloses the mensuration reagent of using the preparation of enzyme reaction product oxidized form nicotinoyl ammonia coenzyme.The enzymic measuring reagent of this invention indication does not add assaying reaction required reduced form nicotinoyl ammonia coenzyme or its analogue, and add its reaction product oxidized form nicotinoyl ammonia coenzyme or its analogue, and generate required enzyme of reduced coenzyme and substrate system accordingly, by in reagent, forming long response time systems such as enzyme-substrate-NAD or enzyme-substrate-NADP, this class oxidized coenzyme or the slow circulation of its analogue are reduced to reduced form nicotinoyl ammonia coenzyme or its analogue, when the reduced form nicotinoyl ammonia coenzyme that is reduced or its analogue reached certain concentration, the enzyme process reagent of this invention indication just can reach the purpose of measuring adenosine deaminase in the sample and isozymes activity thereof.This characteristic feature of an invention is to provide an endogenous synthesizing adenosine desaminase to react the adenosine deaminase reagent of needed reduced form nicotinoyl ammonia coenzyme for the test of adenosine deaminase.One of its shortcoming is, this system is in the needed reduced form nicotinoyl of formation reaction ammonia coenzyme, also offset the activity of part adenosine deaminase reagent (reaction of adenosine deaminase coupling glutamate dehydrogenase must be oxidized to reduced form nicotinoyl ammonia coenzyme oxidized form nicotinoyl ammonia coenzyme), caused the accuracy of reagent test to reduce greatly.Two of its shortcoming is that this reagent must react for some time before official testing in advance, so that can produce enough reduced form nicotinoyl ammonia coenzyme, has so just caused the result that slow action cannot save a critical situation, brings very big inconvenience to test.

Summary of the invention

The technical problem to be solved in the present invention is: propose a kind of measuring method that can overcome the activity of adenosine deaminase of above prior art shortcoming, provide simultaneously in order to realize the adenosine deaminase diagnosis reagent kit of this method.Adopt the reagent in this test kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out adenosine deaminase activity determination, and finding speed is fast, accuracy is high, thereby can obtains practical applying.

It is as follows that the present invention measures the method steps of activity of adenosine deaminase:

1), with sample and the reagent mix of mainly forming by adenosine, unit price phosphoric acid salt, Phosphatase, nucleotide, XOD, peroxidase, urase, reduced form chromogen, make it to take place the reaction of following principle:

Adenosine+water Adenosine deaminaseInosine+ammonium ion

Inosine+unit price phosphoric acid salt Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Urate+water+oxygen UraseUrinate former element+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseIndoleamine chromogen or quinone-imine chromogen+water

2), detect the end reaction thing in the speed that predominant wavelength predominant wavelength 400--600nm absorbancy rises, calculate the active size of adenosine deaminase.

Common step 2) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detect the photoabsorption size that predominant wavelength 400--600nm (difference according to the combination of reduced form chromogen is decided) locates directly to reflect indoleamine chromogen (Indamine dye) or quinone-imine chromogen (Quioneimine dye) content height, draw the adenosine deaminase measurement result.

The blending ratio of above sample and reagent by volume is 1/10 to 1/250, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction times was controlled at conventional 2-30 minute.

The present invention passes through adenosine deaminase, Phosphatase, nucleotide, XOD, urase, a series of enzyme linked reaction such as peroxidase, the most colourless reduced form chromogen combination is oxidized to colored indoleamine chromogen or quinone-imine chromogen, thereby can be by ultraviolet analyser or half, under the automatic clinical chemistry analyzer device, detect predominant wavelength 400--600nm place, measure the height of indoleamine chromogen or quinone-imine chromogen content, and then directly reflect the size of activity of adenosine deaminase, for the diagnosis of multiple disease is provided convenience, whole mensuration process only need add reagent set by step and get final product, accomplish without any letup, be convenient to operation.Compared with prior art, the present invention is not only easy to utilize, and reagent react generates two times of hydrogen peroxide, and sensitivity improves two times in theory.And because the indoleamine chromogen or the quinone-imine chromogen that are generated all have higher molar extinction coefficient, therefore highly sensitive, tolerance range good.

Adenosine deaminase diagnosis reagent kit in order to realization the inventive method can be single agent, comprising:

Damping fluid 40--200mmol/l

Adenosine 1--50mmol/l

Unit price phosphoric acid salt 0.2--10mmol/l

Phosphatase, nucleotide 500--50000U/l

XOD 500--50000U/l

Peroxidase 500--50000U/l

Urase 500--50000U/l

Stablizer 10--80% (cumulative volume)

Reduced form chromogen combination 0.1--20mmol/l

Reduced form chromogen combination can be 3-methyl-2-Proxel hydrazone (3-methyl-2-benzothiazolinone-hydrszone is called for short MBTH) of 0.5-20mmol/l or the amino anti-arsenic of 4-of 0.5-20mmol/l (4-aminoantipyrine AAP) combines with one of 14 kinds of compositions below the 0.1--20mmol/l:

PHENOL 99.8 MIN ((CARBOLIC ACID)) (phenol)

N-ethyl-N-(3-thiopropyl)-m-thialdine amine (N-ethyl-N-(3-sulfopropyl)-m-anisidine is called for short ESPAS)

N, and the two ethyls of N--m-Tolylamine (N, N-Diethyl-m-toluidine)

2, and the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-(2,4-Dichloriphenol)

2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid (2,4,6-Tribromo-3-hydroxy-benzenesulfonic acid is called for short TBHB)

3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid (3,5-Dichlorophenol sulfonic acid) of 5-

3, the two chloro-2-hydroxyl-Phenylsulfonic acids (3,5-Dichloro-2-hydroxy-benzenesulfonic acid is called for short DHBS) of 5-

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine-sodium is called for short TOOS)

Three bromine hydroxy-benzoic acid (Tribromohydroxybenzoic acid)

Two monomethylanilines (Dimethylaniline is called for short DMA)

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine is called for short EHSPT)

2,2 '-AZINO-two (3-ethylbenzene thiazoles-6-sulfonic acid) (2,2 '-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) is called for short ABTS

Vanillic acid (Vanillic acid (4-hydroxy-3-methoxybenzoic acid) is called for short HMB)

3-methyl-ethyl-hydroxyanilines (3-methyl-ethyl-hydroxyaniline is called for short MEHA)

Also above single agent can be made into following pair of agent, more help eliminating the pollution of inside and outside source inosine, xanthoglobulin and uric acid:

Reagent I

Damping fluid 40--200mmol/l

Unit price phosphoric acid salt 0.2--10mmol/l

Phosphatase, nucleotide 500--50000U/l

XOD 500--50000U/l

Peroxidase 500--50000U/l

Urase 500--50000U/l

Stablizer 10--80% (cumulative volume)

One of reduced form chromogen combination 0.1--20mmol/l

Reduced form chromogen combination can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.5-20mmol/l or 0.5-20mmol/l is one of in the two.

Reagent II

Damping fluid 40--200mmol/l

Adenosine 1--50mmol/l

Stablizer 10--80% (cumulative volume)

Two 0.1--20mmol/l of reduced form chromogen combination

Two of reduced form chromogen combination can be one of following 14 kinds of compositions of 0.1--20mmol/l: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

The prescription of two agent is not limited only to above-mentioned prescription, one of reduced form chromogen combination and two can transposition.The composition of reagent I wherein, peroxidase can be placed on reagent II, so can form multiple formulations, does not just describe in detail one by one at this.

Reagent can also be made into following three reagent, not only more help eliminating the pollution of inside and outside source inosine, xanthoglobulin and uric acid, also more help the stable of reagent:

Reagent I

Damping fluid 40--200mmol/l

Unit price phosphoric acid salt 0.2--10mmol/l

Stablizer 10--80% (cumulative volume)

One of reduced form chromogen combination 0.1--20mmol/l

Reduced form chromogen combination can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.5-20mmol/l or 0.5-20mmol/l is one of in the two.

Reagent II

Phosphatase, nucleotide 500--50000U/l

XOD 500--50000U/l

Peroxidase 500--50000U/l

Urase 500--50000U/l

Stablizer 10--80% (cumulative volume)

Reagent III

Damping fluid 40--200mmol/l

Adenosine 1--50mmol/l

Stablizer 10--80% (cumulative volume)

The 0.1--20mmol/l of reduced form chromogen combination

Two of reduced form chromogen combination can be one of following 14 kinds of compositions of 0.1--20mmol/l: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

Three doses prescription is not limited only to above-mentioned prescription, one of reduced form chromogen combination and two can divide any position that is opened in reagent I, II or III.The composition of reagent I wherein, unit price phosphoric acid salt can be placed among the reagent II, the composition of reagent II, Phosphatase, nucleotide, XOD, peroxidase, urase, etc. also can be placed among the reagent I, peroxidase can also be placed on reagent III, so can form multiple formulations, just not describe in detail one by one at this.

The pH scope of buffer reagent can be 6.5-10.5.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphoric acid salt (Phosphate) damping fluid, trolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid, imidazoles (Imidazole) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.

In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer 10-80% among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stablizer can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine, salt or the adenosine diphosphate (ADP) etc.

Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the adenosine deaminase diagnosis reagent kit of the present invention of following system component relation is comparatively desirable:

Damping fluid 80--120mmol/l

Adenosine 1--5mmol/l

Unit price phosphoric acid salt 2--5mmol/l

Phosphatase, nucleotide 5000--50000U/l

XOD 5000--50000U/l

Peroxidase 5000--50000U/l

Urase 5000--50000U/l

Stablizer 30--50% (cumulative volume)

Reduced form chromogen combination 0.5--2mmol/l

The combination of reduced form chromogen can be that 3-methyl-2-Proxel hydrazone of 0.5-2mmol/l or the amino anti-arsenic of 4-and one of 14 kinds of compositions below the 0.5--2mmol/l of 0.5-20mmol/l combine: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

Because the present invention utilizes Enzymology method, enzyme digestion reaction to have the high characteristics of specificity fully, is not vulnerable to the interference of other materials of inside and outside source.Easy, the easy handling of enzyme process method.The specificity of enzyme digestion reaction impels test result accurate.Enzyme digestion reaction all is to carry out under buffer conditions, does not have environmental pollution problem.The enzyme process method does not need special, extra instrument, and testing cost is cheap.Therefore can guarantee to have higher test accuracy, be more convenient for applying.

In addition, one of striking features of the present invention is the pollution that can eliminate inside and outside source inosine, xanthoglobulin and uric acid, the effect of eliminating inside and outside source inosine, xanthoglobulin and uric acid occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source inosine, xanthoglobulin and uric acid, and all be the activity that results from adenosine deaminase at the needed inosine of second half section time test activity of adenosine deaminase.

Embodiment

The present invention is further illustrated below in conjunction with examples of implementation.

Embodiment one (single agent)

The adenosine deaminase diagnosis reagent kit of present embodiment comprises:

Damping fluid 80mmol/l

Adenosine 1mmol/l

Unit price phosphoric acid salt 2mmol/l

Phosphatase, nucleotide 5000U/l

XOD 5000U/l

Peroxidase 5000U/l

Urase 5000U/l

The amino anti-arsenic 2mmol/l of 4-

PHENOL 99.8 MIN ((CARBOLIC ACID)) 10mmol/l

Stablizer 30% (cumulative volume)

On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 495～505nm, more than the test commplementary wave length 600nm, the volume ratio of sample and reagent is 1/25, and the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

After adding sample and reagent, make it to mix, following reaction take place:

Adenosine+water Adenosine deaminaseInosine+ammonium ion

Inosine+unit price phosphoric acid salt Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Urate+water+oxygen UraseUrinate former element+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseIndoleamine chromogen or quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 495～505nm absorbancy rises, thereby calculate the active size of adenosine deaminase.

The present invention passes through adenosine deaminase, Phosphatase, nucleotide, XOD, urase, a series of enzyme linked reaction such as peroxidase, the most colourless reduced form chromogen combination is oxidized to colored indoleamine chromogen or quinone-imine chromogen, thereby can be by ultraviolet analyser or half, under the automatic clinical chemistry analyzer device, detect predominant wavelength 400--600nm place, measure the height of indoleamine chromogen or quinone-imine chromogen content, and then directly reflect the size of activity of adenosine deaminase, for the diagnosis of multiple disease is provided convenience, whole mensuration process only need add reagent set by step and get final product, accomplish without any letup, be convenient to operation.Compared with prior art, the present invention is not only easy to utilize, and reagent react generates two times of hydrogen peroxide, and sensitivity improves two times in theory.And because the indoleamine chromogen or the quinone-imine chromogen that are generated all have higher molar extinction coefficient, therefore highly sensitive, tolerance range good.

Embodiment two (two agent)

The adenosine deaminase diagnosing reagent of present embodiment has:

Reagent I

Damping fluid 100mmol/l

Unit price phosphoric acid salt 3mmol/l

Phosphatase, nucleotide 20000U/l

XOD 20000U/l

Peroxidase 20000U/l

Urase 20000U/l

Stablizer 40% (cumulative volume)

The amino anti-arsenic 1mmol/l of 4-

Reagent II

Damping fluid 100mmol/l

Adenosine 3mmol/l

Stablizer 30mmol/l

2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid 1mmol/l

When measuring activity of adenosine deaminase, temperature is controlled at 30 ℃, 10 minutes reaction times, test predominant wavelength 546nm, more than the test commplementary wave length 630nm, the volume ratio of sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

Concrete determination step is:

Adenosine+water Adenosine deaminaseInosine+ammonium ion

Inosine+unit price phosphoric acid salt Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Urate+water+oxygen UraseUrinate former element+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseIndoleamine chromogen or quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 500～600nm luminosity rises, thereby calculate the active size of adenosine deaminase.

The reaction times of each reactions steps was controlled at 10 minutes.

Embodiment three (three doses)

The adenosine deaminase diagnosing reagent of present embodiment is three doses, has:

Reagent I

Damping fluid 120mmol/l

Unit price phosphoric acid salt 5mmol/l

Stablizer 25mmol/l

3-methyl-2-Proxel hydrazone 2mmol/l

Reagent II

Damping fluid 120mmol/l

Phosphatase, nucleotide 50000U/l

XOD 50000U/l

Peroxidase 50000U/l

Urase 50000U/l

Stablizer 50% (cumulative volume)

Reagent III

Damping fluid 120mmol/l

Adenosine 5mmol/l

Two monomethylaniline 2mmol/l

Stablizer 30mmol/l

On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, the volume ratio of sample and reagent is 1/25, and the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

Concrete determination step is:

Adenosine+water Adenosine deaminaseInosine+ammonium ion

Inosine+unit price phosphoric acid salt Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Urate+water+oxygen UraseUrinate former element+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseIndoleamine chromogen or quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 578nm absorbancy rises, thereby calculate the active size of adenosine deaminase.

The reaction times of each reactions steps was controlled at 10 minutes.

Embodiment four

The adenosine deaminase diagnosing reagent of present embodiment has:

Reagent I

Damping fluid 100mmol/l

Unit price phosphoric acid salt 4mmol/l

Phosphatase, nucleotide 10000U/l

XOD 10000U/l

Peroxidase 10000U/l

Urase 10000U/l

Stablizer 50% (cumulative volume)

The amino anti-arsenic 1mmol/l of 4-

Reagent II

Damping fluid 100mmol/l

Adenosine 2mmol/l

Stablizer 30mmol/l

PHENOL 99.8 MIN ((CARBOLIC ACID)) 7mmol/l

On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 500nm, more than the test commplementary wave length 600nm, the volume ratio of sample and reagent is 1/25, the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times.

After adding sample and reagent, make it to mix, following reaction take place:

Adenosine+water Adenosine deaminaseInosine+ammonium ion

Inosine+unit price phosphoric acid salt Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Urate+water+oxygen UraseUrinate former element+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseIndoleamine chromogen or quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 500nm absorbancy rises, thereby calculate the active size of adenosine deaminase.

In a word, experiment showed, and adopt measuring method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, tolerance range good, is not subjected to the pollution of inside and outside source material.

Claims (10)

1. method of measuring activity of adenosine deaminase, step is as follows:

1), with sample and the reagent mix of mainly forming by adenosine, unit price phosphoric acid salt, Phosphatase, nucleotide, XOD, peroxidase, urase, reduced form chromogen, make it to take place following reaction:

Adenosine+water Adenosine deaminaseInosine+ammonium ion

Inosine+unit price phosphoric acid salt Phosphatase, nucleotideXanthoglobulin+ribose 1-phosphate

Xanthoglobulin+water+oxygen XODUrate+hydrogen peroxide

Urate+water+oxygen UraseUrinate former element+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseIndoleamine chromogen or quinone Asia

Amine chromogen+water

2), detecting the end reaction thing in predominant wavelength predominant wavelength 400---the speed that the 600nm absorbancy rises, the activity that calculates adenosine deaminase is big or small.

2. according to the method for the described mensuration activity of adenosine deaminase of claim 1, it is characterized in that: described step 2) be, the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect predominant wavelength 400-600nm, more than the test commplementary wave length 500-700nm, the speed that absorbancy rises calculates the active big or small of adenosine deaminase.

3, according to the method for claim 1 or 2 described mensuration activity of adenosine deaminase, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction times was controlled at 2-30 minute.

4, according to the method for claim 1 or 2 described mensuration activity of adenosine deaminase, it is characterized in that: the ratio control of sample and reagent is 1/10 to 1/250.

5. adenosine deaminase diagnosis reagent kit comprises:

Damping fluid 40---200mmol/l

Adenosine 1---50mmol/l

Unit price phosphoric acid salt 0.2---10mmol/l

Phosphatase, nucleotide 500---50000U/l

XOD 500---50000U/l

Peroxidase 500---50000U/l

Urase 500---50000U/l

Stablizer 10---80% (cumulative volume)

Reduced form chromogen combination 0.1---20mmol/l

6. according to the described adenosine deaminase diagnosis reagent kit of claim 5, it is characterized in that: reagent is made into single agent, two agent or three doses.

7. according to claim 5 or 6 described adenosine deaminase diagnosis reagent kits, it is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, ammonium sulfate, salt or the adenosine diphosphate (ADP).

8. according to claim 5 or 6 described adenosine deaminase diagnosis reagent kits, it is characterized in that: described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, phosphate buffered saline buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid, and the pH scope is 6.5-10.5.

9, according to claim 5 or 6 described adenosine deaminase diagnosis reagent kits, it is characterized in that: one of following 14 kinds of compositions of 3-methyl-2-Proxel hydrazone that described reduced form chromogen is 0.1-20mmol/l and 0.1-20mmol/l combine:

PHENOL 99.8 MIN ((CARBOLIC ACID))

N-ethyl-N-(3-thiopropyl)-m-thialdine amine

N, the two ethyls of N--m-Tolylamine

2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-

2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid

3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-

3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt

Three bromine hydroxy-benzoic acid

Two monomethylanilines

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine

2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid)

Vanillic acid

3-methyl-ethyl-hydroxyanilines.

10, according to claim 5 or 6 described adenosine deaminase diagnosing reagents, it is characterized in that: described reduced form chromogen is combined as by the amino anti-arsenic of the 4-of 0.1-20mmol/l and one of 14 kinds of compositions below the 0.1-20mmol/l and combines:

PHENOL 99.8 MIN ((CARBOLIC ACID))

N-ethyl-N-(3-thiopropyl)-m-thialdine amine

N, the two ethyls of N--m-Tolylamine

2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-

2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid

3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-

3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt

Three bromine hydroxy-benzoic acid

Two monomethylanilines

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine

2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid)

Vanillic acid

3-methyl-ethyl-hydroxyanilines.