CN1769475A - Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit - Google Patents
Adenosine deaminase activity determination method and adenosine deaminase diagnosi kit Download PDFInfo
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- CN1769475A CN1769475A CN 200410065605 CN200410065605A CN1769475A CN 1769475 A CN1769475 A CN 1769475A CN 200410065605 CN200410065605 CN 200410065605 CN 200410065605 A CN200410065605 A CN 200410065605A CN 1769475 A CN1769475 A CN 1769475A
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- acid
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- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 title claims abstract description 62
- 102000055025 Adenosine deaminases Human genes 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 32
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- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 79
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 43
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- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims abstract description 21
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for determining the activity of adenosine deaminase, and also the reagent kit for adenosine deaminase diagnosis. The reagent kit comprises cushioning solution, adenosine, adenosine triphosphate, glutacid, pyroracemic acid, glutamine synthetase, pyruvic oxidase, peroxydase, deacidized chromogen combination, and stabilizer. By mixing sample and reagent of a predetermiend volumetric ratio, generating coupling reaction between them, subjecting the final reactant to biochemiscal analyser, the main wavelength absorbancy variance ratio (speed) can be detected, and the activity of the adenosine deaminase can thus be measured. The method of the invention can be used to obtain the needed measurement result purely through biochemical analytic instruments, and advantages of the method include higher sensibility, better accuracy, less susceptibility to contamination of internal or external materials, and easy application.
Description
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, adenosine triphosphate, L-glutamic acid, pyruvic acid, glutamine synthetase, pyruvic oxidase, peroxidase, make it to take place the reaction of following principle:
Adenosine+water
Adenosine deaminaseInosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+phosphate radical+glutamine
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+carbonic acid gas+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 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 uses enzyme linked reaction and reduced form chromogen (Chromogen) combined system reaction continuous monitoring methods such as adenosine deaminase coupling glutamine synthetase, pyruvic oxidase, peroxidase.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling glutamine synthetase again, generate phosphate radical with adenosine triphosphate and L-glutamic acid, phosphate radical produces hydrogen peroxide by the effect of coupling pyruvic oxidase again, hydrogen peroxide is under the superoxide enzyme catalysis, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen (Quioneimine) or indoleamine chromogen (Indamine) dyestuff, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect the size of activity of adenosine deaminase.The advantage of this enzyme linked reaction system has: colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen in (1) its utilization or indoleamine chromogen dyestuff reflects activity of adenosine deaminase, and the good advantage of tolerance range is arranged.(2) reacted constituent of this enzyme linked reaction system composition all adds, and is not subjected to the pollution of inside and outside source material, and test result is accurate.
The adenosine deaminase diagnosis reagent kit of realizing the inventive method can be single agent, comprising:
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Adenosine triphosphate 1--10mmol/l
L-glutamic acid 1--30mmol/l
Pyruvic acid 1--20mmol/l
Glutamine synthetase 1000--50000U/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--50000U/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination 0.1--20mmol/l
3-methyl-2-Proxel hydrazone (3-methyl-2-benzothiazolinone-hydrszone is called for short MBTH) that above reduced form chromogen combination can be 0.1-20mmol/l or the amino anti-arsenic of 4-(4-aminoantipyrine) of 0.1-20mmol/l combine with one of following 14 kinds of compositions of 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-Dichlorophenolsulfonic acid) of 5-
3, the two chloro-2-hydroxyl-Phenylsulfonic acids (3,5-Dichloro-2-hydroxy-benzenesulfonicacid 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 following pair of agent be can be made into, inside and outside source ammonia, phosphate radical pollution more helped eliminating:
Reagent I
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
L-glutamic acid 1--30mmol/l
Pyruvic acid 1--20mmol/l
Glutamine synthetase 1000--50000U/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--50000U/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination (one) 0.1--20mmol/l
Reduced form chromogen combination (one) can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.1-20mmol/l or 0.1-20mmol/l is one of in the two.
Reagent II
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination (two) 0.1--20mmol/l
Reduced form chromogen combination (two) 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, and reduced form chromogen combination () and (two) can transpositions.The composition of reagent I wherein, adenosine triphosphate, L-glutamic acid, pyruvic acid, glutamine synthetase, pyruvic oxidase, peroxidase etc. can be placed on reagent II, and reagent II composition wherein, adenosine also can be placed on reagent I, so can form multiple formulations, just not describe in detail one by one at this.
Reagent can also be made into following three reagent, not only more help eliminating inside and outside source ammonia, phosphate radical pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Adenosine triphosphate 1--10mmol/l
L-glutamic acid 1--30mmol/l
Pyruvic acid 1--20mmol/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination (one) 0.1--20mmol/l
Reduced form chromogen combination (one) can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.1-20mmol/l or 0.1-20mmol/l is one of in the two.
Reagent II
Damping fluid 40--200mmol/l
Glutamine synthetase 1000--50000U/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--50000U/l
Stablizer 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Stablizer 10--80% (cumulative volume)
Reduced form chromogen combination (two) 0.1--20mmol/l
Reduced form chromogen combination (two) 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, and 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, adenosine triphosphate, L-glutamic acid, pyruvic acid etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, glutamine synthetase, pyruvic oxidase, peroxidase etc. also can be placed among reagent I or the reagent III.Reagent III composition wherein, adenosine also can be placed among reagent I or the reagent II, so can form multiple formulations, do not describe in detail one by one at this.
The pH scope of buffer reagent can be 6.0～11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) 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% or 10～50mmol/l 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 or the salt 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 2--10mmol/l
Adenosine triphosphate 2--8mmol/l
L-glutamic acid 5--10mmol/l
Pyruvic acid 1--10mmol/l
Glutamine synthetase 4000--10000U/l
Pyruvic oxidase 5000--20000U/l
Peroxidase 5000--20000U/l
Stablizer 20--50% (cumulative volume)
Reduced form chromogen combination 0.5--10mmol/l
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 ammonia, phosphate radical, the effect of eliminating inside and outside source ammonia, phosphate radical occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source ammonia, phosphate radical, and all be the activity that results from adenosine deaminase at the needed ammonia of second half section time test activity of adenosine deaminase, phosphate radical.
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 2mmol/l
Adenosine triphosphate 2mmol/l
L-glutamic acid 5mmol/l
Pyruvic acid 1mmol/l
Glutamine synthetase 4000U/l
Pyruvic oxidase 5000U/l
Peroxidase 5000U/l
Stablizer 50% (cumulative volume)
The amino anti-arsenic 2mmol/l of 4-
PHENOL 99.8 MIN ((CARBOLIC ACID)) 10mmol/l
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 tested adenosine deaminase 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
Adenosine triphosphate+L-glutamic acid+ammonia
Glutamine synthetaseAdenosine diphosphate (ADP)+phosphate radical+glutamine
Phosphate radical+pyruvic acid+oxygen+water
Pyruvic oxidaseAcetylphosphate+carbonic acid gas+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.
Present embodiment is used enzyme linked reaction and reduced form chromogen (Chromogen) combined system reaction continuous monitoring methods such as adenosine deaminase coupling glutamine synthetase, pyruvic oxidase, peroxidase.Adenosine deaminase enzymolysis adenosine produces ammonia, effect by the coupling glutamine synthetase again, generate phosphate radical with adenosine triphosphate and L-glutamic acid, phosphate radical produces hydrogen peroxide by the effect of coupling pyruvic oxidase again, hydrogen peroxide is under the superoxide enzyme catalysis, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen (Quioneimine) or indoleamine chromogen (Indamine) dyestuff, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect the size of activity of adenosine deaminase.
Embodiment two (two agent)
The adenosine deaminase diagnosing reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Adenosine triphosphate 5mmol/l
L-glutamic acid 8mmol/l
Pyruvic acid 6mmol/l
Glutamine synthetase 7000U/l
Pyruvic oxidase 12000U/l
Peroxidase 12000U/l
Stablizer 50% (cumulative volume)
The amino anti-arsenic 1mmol/l of 4-
Reagent II
Damping fluid 100mmol/l
Adenosine 6mmol/l
Stablizer 20mmol/l
2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid 1mmol/l
When measuring activity of adenosine deaminase, temperature is controlled at 30 ℃, 15 minutes reaction times, test predominant wavelength 546nm, more than the test commplementary wave length 600nm, the volume ratio of tested adenosine deaminase 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 gland guanosine deaminase inosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia glutamine synthetase adenosine diphosphate (ADP)+phosphate radical+glutamine
Phosphate radical+pyruvic acid+oxygen+water pyruvic oxidase acetylphosphate+carbonic acid gas+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination peroxidase indoleamine chromogen or quinone-imine chromogen+water
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 546nm absorbancy rises, thereby calculate the active size of adenosine deaminase.
The reaction times of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
The adenosine deaminase diagnosing reagent of present embodiment is three doses, has:
Reagent I
Damping fluid 120mmol/l
Adenosine triphosphate 8mmol/l
L-glutamic acid 10mmol/l
Pyruvic acid 10mmol/l
Stablizer 20mmol/l
3-methyl-2-Proxel hydrazone 2mmol/l
Reagent II
Damping fluid 120mmol/l
Glutamine synthetase 10000U/l
Pyruvic oxidase 20000U/l
Peroxidase 20000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Adenosine 10mmol/l
Stablizer 20mmol/l
Two monomethylaniline 2mmol/l
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, the volume ratio of tested adenosine deaminase 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 gland guanosine deaminase inosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia glutamine synthetase adenosine diphosphate (ADP)+phosphate radical+glutamine
Phosphate radical+pyruvic acid+oxygen+water pyruvic oxidase acetylphosphate+carbonic acid gas+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination peroxidase indoleamine 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.
Embodiment four
The adenosine deaminase diagnosing reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Adenosine triphosphate 2mmol/l
L-glutamic acid 10mmol/l
Pyruvic acid 10mmol/l
Stablizer 20mmol/l
The amino anti-arsenic 1mmol/l of 4-
Reagent II
Damping fluid 100mmol/l
Adenosine 3mmol/l
Glutamine synthetase 6000U/l
Pyruvic oxidase 10000U/l
Peroxidase 20000U/l
Stablizer 50% (cumulative volume)
Two monomethylaniline 2mmol/l
When measuring activity of adenosine deaminase, temperature is controlled at 30 ℃, 10 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, the volume ratio of tested adenosine deaminase 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 gland guanosine deaminase inosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia glutamine synthetase adenosine diphosphate (ADP)+phosphate radical+glutamine
Phosphate radical+pyruvic acid+oxygen+water pyruvic oxidase acetylphosphate+carbonic acid gas+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination peroxidase indoleamine 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 5 minutes.
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, adenosine triphosphate, L-glutamic acid, pyruvic acid, glutamine synthetase, pyruvic oxidase, peroxidase, make it to take place the reaction of following principle:
Adenosine+water gland guanosine deaminase inosine+ammonium ion
Adenosine triphosphate+L-glutamic acid+ammonia glutamine synthetase adenosine diphosphate (ADP)+
Phosphate radical+glutamine
Phosphate radical+pyruvic acid+oxygen+water pyruvic oxidase acetylphosphate+
Carbonic acid gas+hydrogen peroxide
Hydrogen peroxide+reduced form chromogen combination peroxidase indoleamine chromogen or
Quinone-imine chromogen+water
2), detect the end reaction thing in the speed that predominant wavelength 400-600nm absorbancy rises, calculate the active size of adenosine deaminase.
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, 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 is grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Adenosine 0.5--50mmol/l
Adenosine triphosphate 1--10mmol/l
L-glutamic acid 1--30mmol/l
Pyruvic acid 1--20mmol/l
Glutamine synthetase 1000--50000U/l
Pyruvic oxidase 5000--50000U/l
Peroxidase 5000--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: described 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 or the salt.
8. according to claim 5 or 6 described adenosine deaminase diagnosis reagent kits, it is characterized in that: the pH scope of described buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.
9, according to claim 5 or 6 described adenosine deaminase diagnosing reagents, 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.
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| EP3279658A1 (en) * | 2016-08-05 | 2018-02-07 | ARKRAY, Inc. | Quantification method for ammonia, quantification reagent, quantification reagent kit, and ammonia quantification device |
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