CN1746316A - Determination of adenosine deaminase activity and diagnostic kit of adenosine deaminase - Google Patents
Determination of adenosine deaminase activity and diagnostic kit of adenosine deaminase Download PDFInfo
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- CN1746316A CN1746316A CN 200410041906 CN200410041906A CN1746316A CN 1746316 A CN1746316 A CN 1746316A CN 200410041906 CN200410041906 CN 200410041906 CN 200410041906 A CN200410041906 A CN 200410041906A CN 1746316 A CN1746316 A CN 1746316A
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- adenosine deaminase
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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, ethanol, peroxidase, oxidized coenzyme, aldehyde dehydrogenase 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
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 (H
2O)
Adenosine deaminaseInosine (Inosine)+ammonium ion (NH
4 +), 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, ethanol, peroxidase, oxidized coenzyme, aldehyde dehydrogenase, 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
Hydrogen peroxide+ethanol
PeroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water aldehyde dehydrogenase acetate+reducibility coenzyme+hydrogen ion
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbancy rises, calculate the active size of adenosine deaminase.
Common step 2) is that the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detects the speed that predominant wavelength 340nm absorbancy rises, to calculate the active big or small of adenosine deaminase.
The blending ratio of above sample and reagent by volume is 1/10 to 1/250, and above process is measured temperature and is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction times was controlled at conventional 2-30 minute.
This method is used adenosine deaminase coupling Phosphatase, nucleotide and is generated xanthoglobulin, coupling XOD again generates urate and hydrogen peroxide with the xanthoglobulin oxidation, coupling generates acetaldehyde through the superoxide enzyme reaction again, under the help of oxidized coenzyme, aldehyde dehydrogenase becomes acetate with oxidation of acetaldehyde, simultaneous oxidation type coenzyme is reduced into reducibility coenzyme, reducibility coenzyme has absorption peak at wavelength 340nm place, therefore from measuring the speed that predominant wavelength 340nm absorbancy rises, can calculate the active size of adenosine deaminase.
Adenosine deaminase diagnosis reagent kit in order to realization the inventive method can be single agent, comprising:
Buffer reagent 40--200mmol/l
Adenosine 0.2--20mmol/l
Unit price phosphoric acid salt 0.2--10mmol/l
Phosphatase, nucleotide 500--50000U/l
XOD 500--50000U/l
Ethanol 1--30mmol/l
Peroxidase 500--50000U/l
Oxidized coenzyme 0.5--20mmol/l
Aldehyde dehydrogenase 500--50000U/l
Stablizer 10--50% (cumulative volume)
Also above single agent can be made into following pair of agent, more help eliminating endogenous inosine, reach hypoxanthic pollution:
Reagent I
Damping fluid 40--200mmol/l
Unit price phosphoric acid salt 0.2--10mmol/l
Ethanol 1--30mmol/l
Oxidized coenzyme 0.5--20mmol/l
Phosphatase, nucleotide 500--50000U/l
XOD 500--50000U/l
Peroxidase 500--50000U/l
Aldehyde dehydrogenase 500--50000U/l
Stablizer 10--50% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Adenosine 0.2--20mmol/l
Stablizer 10--50% (cumulative volume)
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, and ethanol, oxidized coenzyme, peroxidase and aldehyde dehydrogenase etc. can be placed on reagent II, so can form multiple formulations, do not describe in detail one by one at this.
Reagent can also be made into following three reagent, not only more help eliminating inosine and hypoxanthic pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Unit price phosphoric acid salt 0.2--10mmol/l
Ethanol 1--30mmol/l
Oxidized coenzyme 0.5--20mmol/l
Stablizer 10--50mmol/l
Reagent II
Phosphatase, nucleotide 500--50000U/l
XOD 500--50000U/l
Peroxidase 500--50000U/l
Aldehyde dehydrogenase 500--50000U/l
Stablizer 10--50% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Adenosine 0.2--20mmol/l
Stablizer 10--50mmol/l
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, and ethanol, oxidized coenzyme etc. can be placed among reagent II or the reagent III individually or simultaneously, and unit price phosphoric acid salt can be placed among the reagent II; Composition among the reagent II, any independent or any combination also can be placed on reagent I in Phosphatase, nucleotide, XOD, peroxidase and the aldehyde dehydrogenase etc.; In peroxidase and the aldehyde dehydrogenase etc. separately or the two can be placed among the reagent III, so can form multiple formulations, do not describe in detail one by one at this.
The pH scope of buffer reagent is 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.
Above oxidized coenzyme can be reduced form nicotinamide coenzyme or derivatives thereofs such as NADP+, NAD+ or thio-NAD+.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually adding stablizer among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III is the 10-80% (perhaps 10--50mmol/l) of cumulative volume, and 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--10mmol/l
Phosphatase, nucleotide 5000--20000U/l
XOD 5000--20000U/l
Ethanol 5--10mmol/l
Peroxidase 10000--20000U/l
Oxidized coenzyme 1--2mmol/l
Aldehyde dehydrogenase 10000--20000U/l
Because the present invention utilizes Enzymology method fully, enzyme digestion reaction has the high characteristics of specificity, 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 endogenous inosine, xanthoglobulin and hydrogen peroxide, the effect of eliminating endogenous inosine, xanthoglobulin and hydrogen peroxide occurs in the first half of entire reaction time period, be consumed totally at contaminated endogenous inosine of time second half section, xanthoglobulin and hydrogen peroxide, 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 100mmol/l
Adenosine 1mmol/l
Unit price phosphoric acid salt 2mmol/l
Phosphatase, nucleotide 10000U/l
XOD 10000U/l
Ethanol 5mmol/l
Peroxidase 20000U/l
Oxidized coenzyme 1mmol/l
Aldehyde dehydrogenase 10000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, 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, theoretical k value-4180.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine+water gland guanosine deaminase inosine+ammonium ion
Inosine+unit price phosphoric acid salt Phosphatase, nucleotide xanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen XOD urate+hydrogen peroxide
Hydrogen peroxide+ethanol peroxidase acetaldehyde+water
Acetaldehyde+oxidized coenzyme+water aldehyde dehydrogenase acetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy rises, thereby calculate the active size of adenosine deaminase.
Present embodiment is used adenosine deaminase coupling Phosphatase, nucleotide and is generated xanthoglobulin, coupling XOD again generates urate and hydrogen peroxide with the xanthoglobulin oxidation, coupling generates acetaldehyde through the superoxide enzyme reaction again, under the help of oxidized coenzyme, aldehyde dehydrogenase becomes acetate with oxidation of acetaldehyde, simultaneous oxidation type coenzyme is reduced into reducibility coenzyme, reducibility coenzyme has absorption peak at wavelength 340nm place, therefore from measuring the speed that predominant wavelength 340nm absorbancy rises, can calculate the active size of adenosine deaminase.
Embodiment two (two agent)
The adenosine deaminase diagnosing reagent of present embodiment has:
Reagent I
Damping fluid 120mmol/l
Unit price phosphoric acid salt 8mmol/l
Ethanol 8mmol/l
Oxidized coenzyme 2mmol/l
Phosphatase, nucleotide 20000U/l
XOD 20000U/l
Peroxidase 10000U/l
Aldehyde dehydrogenase 20000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 120mmol/l
Adenosine 5mmol/l
Stablizer 50mmol/l
When measuring activity of adenosine deaminase, temperature is controlled at 30 ℃, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, 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, theoretical k value-4180.
Concrete determination step is:
Adenosine+water gland guanosine deaminase inosine+ammonium ion
Inosine+unit price phosphoric acid salt Phosphatase, nucleotide xanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen XOD urate+hydrogen peroxide
Hydrogen peroxide+ethanol peroxidase acetaldehyde+water
Acetaldehyde+oxidized coenzyme+water aldehyde dehydrogenase acetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy 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 80mmol/l
Unit price phosphoric acid salt 5mmol/l
Ethanol 7mmol/l
Oxidized coenzyme 2mmol/l
Stablizer 30mmol/l
Reagent II
Damping fluid 80mmol/l
Phosphatase, nucleotide 5000U/l
XOD 5000U/l
Peroxidase 15000U/l
Aldehyde dehydrogenase 15000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 80mmol/l
Adenosine 3mmol/l
Stablizer 20mmol/l
On automatic clinical chemistry analyzer, set: 30 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, 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, theoretical k value-4180.
Concrete determination step is:
Adenosine+water gland guanosine deaminase inosine+ammonium ion
Inosine+unit price phosphoric acid salt Phosphatase, nucleotide xanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen XOD urate+hydrogen peroxide
Hydrogen peroxide+ethanol peroxidase acetaldehyde+water
Acetaldehyde+oxidized coenzyme+water aldehyde dehydrogenase acetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm 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 120mmol/l
Unit price phosphoric acid salt 6mmol/l
Ethanol 8mmol/l
Oxidized coenzyme 2mmol/l
Phosphatase, nucleotide 15000U/l
XOD 15000U/l
Peroxidase 15000U/l
Aldehyde dehydrogenase 15000U/l
Stablizer 40% (cumulative volume)
Reagent II
Damping fluid 120mmol/l
Adenosine 3mmol/l
Stablizer 30mmol/l
On Biochemical Analyzer, set: 25 ℃ of temperature, 15 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of sample and reagent is 2/25, and the Direction of Reaction is positive reaction, 1 minute time of lag, 2 minutes detection times, theoretical k value-2170.
After adding sample and reagent, make it to mix, following reaction take place:
Adenosine+water gland guanosine deaminase inosine+ammonium ion
Inosine+unit price phosphoric acid salt Phosphatase, nucleotide xanthoglobulin+ribose 1-phosphate
Xanthoglobulin+water+oxygen XOD urate+hydrogen peroxide
Hydrogen peroxide+ethanol peroxidase acetaldehyde+water
Acetaldehyde+oxidized coenzyme+water aldehyde dehydrogenase acetate+reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 340nm absorbancy rises, thereby calculate the active size of adenosine deaminase.
The reaction times of each reactions steps was controlled at 10 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 (9)
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, ethanol, peroxidase, oxidized coenzyme, aldehyde dehydrogenase, 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
Hydrogen peroxide+ethanol
PeroxidaseAcetaldehyde+water
Acetaldehyde+oxidized coenzyme+water
Aldehyde dehydrogenaseAcetate+reducibility coenzyme+hydrogen ion
2), detect the end reaction thing in the speed that predominant wavelength 340nm 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 340nm, more than the test commplementary wave length 405nm, 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 the described mensuration activity of adenosine deaminase of claim 3, 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:
Buffer reagent 40---200mmol/l
Adenosine 0.2---20mmol/l
Unit price phosphoric acid salt 0.2---10mmol/l
Phosphatase, nucleotide 500---50000U/l
XOD 500---50000U/l
Ethanol 1---30mmol/l
Peroxidase 500---50000U/l
Oxidized coenzyme 0.5---20mmol/l
Aldehyde dehydrogenase 500---50000U/l
Stablizer 10---50%
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 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: described oxidized coenzyme is a kind of in NADP+, NAD+, the thio-NAD+ reduced form nicotinamide coenzyme or derivatives thereof.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324630A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit |
| CN101329257B (en) * | 2007-06-21 | 2013-06-19 | 苏州艾杰生物科技有限公司 | Nitrous acid determination reagent kit and method for determining nitric acid concentration |
| CN109112179A (en) * | 2017-06-24 | 2019-01-01 | 浙江亚培生物技术有限公司 | A kind of adenosine deaminase activity determination method and adenosine deaminase detection kit |
-
2004
- 2004-09-08 CN CN 200410041906 patent/CN1746316A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101324630A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | Method for determining ethyl hydrate concentration ethyl hydrate diagnosis reagent kit |
| CN101329257B (en) * | 2007-06-21 | 2013-06-19 | 苏州艾杰生物科技有限公司 | Nitrous acid determination reagent kit and method for determining nitric acid concentration |
| CN109112179A (en) * | 2017-06-24 | 2019-01-01 | 浙江亚培生物技术有限公司 | A kind of adenosine deaminase activity determination method and adenosine deaminase detection kit |
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