CN1760369A - Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase - Google Patents
Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase Download PDFInfo
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- CN1760369A CN1760369A CN 200410064906 CN200410064906A CN1760369A CN 1760369 A CN1760369 A CN 1760369A CN 200410064906 CN200410064906 CN 200410064906 CN 200410064906 A CN200410064906 A CN 200410064906A CN 1760369 A CN1760369 A CN 1760369A
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Abstract
A reagent kit for diagnosing 5'-nucleotidase is composed of buffer liquid, inosine monophosphate, monophosphate, nucleoside phosphatase, xanthine oxidase, alcohol, hydrogen peroxidase, oxidized coenzyme, aldehyde dehydrogenase and stabilizer. A process for measuring the activity of 5'-nucleotidase includes such steps as proportionally mixing specimen with reagents, enzyme coupling reaction and biochemically analyzing the final resultant to determine the variation in light absorptivity of master wavelength and in turn the activity of 5'-nucleotidase. Its advantages are high sensitivity and precision, and no pollution.
Description
Technical Field
The invention relates to a method for measuring 5 '-nucleotidase activity, and also relates to a 5' -nucleotidase diagnostic kit for realizing the method, belonging to the technical field of medical test and measurement.
Background
Medical studies have shown that an increase in 5' -nucleotidase (5NT) is mainly seen in obstructive jaundice, as well as in liver cancer and hepatitis. The 5NT increase rate is higher than that of alkaline phosphatase when cholestasis is accompanied by cholangitis, primary and secondary biliary cirrhosis and chronic hepatitis; the sensitivity of 5NT increase is higher than that of alkaline phosphatase in liver tumor and liver granuloma. Because the activity of 5' -nucleotidase is not physiologically increased, the method is more sensitive and specific than alkaline phosphatase in diagnosing infant liver diseases and pregnancy induced liver function cholestasis. Therefore, determination of the activity of 5' -nucleotidase is of great significance for the diagnosis of diseases.
The activity of 5' -nucleotidase is measured by many methods, including isotope substrate method and chemical strong acid phosphorus measuring method (1925). To the best of the applicant's knowledge, the isotope substrate assay is currently used internationally and generally, the method is: 5' -nucleotidase acting on H3dUMP, and after the reaction was terminated, the 5' -nucleotidase activity was determined by analyzing the results with an ion exchange chromatography column. Or the 5 '-nucleotidase acts on adenosine monophosphate to generate inorganic phosphorus, and the content of the inorganic phosphorus is analyzed by a chemical strong acid method to calculate the activity of the 5' -nucleotidase.
The isotope substrate method is complex, has isotope pollution and needs an isotope analyzer, so that the practical popularization and application are difficult. The inorganic phosphorus determination method invented in 1925 is not good in accuracy, strong acid pollutes environment and the like, and is not suitable for popularization and application.
Disclosure of Invention
A method for measuring the activity of 5 '-nucleotidase which can overcome the above disadvantages of the prior art is provided, and a diagnostic kit for 5' -nucleotidase for carrying out the method is also provided. The reagent in the kit can be used for measuring the activity of the 5' -nucleotidase on an ultraviolet/visible light analyzer or a semi-automatic or full-automatic biochemical analyzer, and has high measuring speed and high accuracy, so that the kit can be practically popularized and applied.
The steps for determining the activity of 5' -nucleotidase in the invention are as follows:
1) mixing the sample with a reagent mainly consisting of inosine monophosphate, nucleoside phosphatase, xanthine oxidase, ethanol, catalase, oxidized coenzyme and aldehyde dehydrogenase, so that the following reaction occurs:
inosine monophosphate + water5' -nucleotidaseInosine + monophosphate
Inosine + monophosphateNucleoside phosphatasesHypoxanthine + 1-ribose phosphate
Hypoxanthine + water + oxygenXanthine oxidaseUrate + hydrogen peroxide
Hydrogen peroxide + ethanolCatalase enzymeAcetaldehyde + water
Acetaldehyde, oxidized coenzyme and waterAldehyde dehydrogenaseAcetic acid + reducing coenzyme + hydrogen ion
2) And detecting the descending speed of the final reactant at the main wavelength of 340nm to measure and calculate the activity of the 5' -nucleotidase.
Generally, in the step 2), the final reactant is placed under an ultraviolet/visible light analyzer or a semi-automatic or full-automatic biochemical analyzer, the velocity of the decrease in absorbance at 340nm of the dominant wavelength is detected, and the activity of the 5' -nucleotidase is measured.
The mixing ratio of the above samples and the reagents is 1/10-1/250 by volume, the measuring temperature of the above processis controlled in the conventional range of 20 ℃ to 50 ℃, and the reaction time is controlled in the conventional range of 2-30 minutes.
The method applies enzyme coupling reaction systems such as 5 '-nucleotidase, nucleoside phosphatase, xanthine oxidase, catalase, aldehyde dehydrogenase and the like to finally reduce oxidized coenzyme into reduced coenzyme, and because the reduced coenzyme has an absorption peak at the wavelength of 340nm, the increase degree of the absorption peak at the wavelength of 340nm can directly reflect the activity of 5' -nucleotidase. The advantages of this enzyme-coupled reaction system are:
it uses the measurement of the amount of the oxidized coenzyme reduced to the reduced coenzyme to reflect the 5' -nucleotidase activity, and the stability of the oxidized coenzyme in solution is much higher than that of the reduced coenzyme, so that the stability of the system is very good.
The reaction components formed by the enzyme coupling reaction system are externally added, so that the enzyme coupling reaction system is not polluted by internal and external substances, and the test result is accurate.
The 5' -nucleotidase diagnostic kit for carrying out the method of the present invention may be a single agent comprising:
buffer 40-200 mmol/l
Inosine monophosphate 1-50 mmol/l
Monophosphate 0.2-10 mmol/l
Nucleoside phosphatase 500-50000U/l
Xanthine oxidase 500-50000U/l
Ethanol 1-30 mmol/l
Catalase 500- -50000U/l
Oxidized coenzyme 0.5-20 mmol/l
Aldehyde dehydrogenase 500- -50000U/l
Stabilizer 10-80%(total volume)
The single preparation can also be prepared into the following double preparations, which is more favorable for eliminating the pollution of internal and external sources of inosine and hypoxanthine:
reagent I
Buffer 40-200 mmol/l
Monophosphate 0.2-10 mmol/l
Nucleoside phosphatase 500-50000U/l
Xanthine oxidase 500-50000U/l
Ethanol 1-30 mmol/l
Catalase 500- -50000U/l
Oxidized coenzyme 0.5-20 mmol/l
Aldehyde dehydrogenase 500- -50000U/l
Stabilizer 10-80% (total volume)
Reagent II
Buffer 40-200 mmol/l
Inosine monophosphate 1-50 mmol/l
Stabilizer 10-50mmol/l
The formulation of the double agent is not limited to the above formulation, and the components of the reagent I, ethanol, catalase, aldehyde dehydrogenase, etc. can be put in the reagent II, so that various formulations can be formed, which are not described in detail herein.
The reagent can be prepared into the following three reagents, which not only is more favorable for eliminating the pollution of internal and external sources inosine and hypoxanthine, but also is more favorable for the stability of the reagent:
reagent I
Buffer 40-200 mmol/l
Monophosphate 0.2-10 mmol/l
Ethanol 1-30 mmol/l
Oxidized coenzyme 0.5-20 mmol/l
Stabilizer 10-50mmol/l
Reagent II
Buffer 40-200 mmol/l
Nucleoside phosphatase 500-50000U/l
Xanthine oxidase 500-50000U/l
Catalase 500- -50000U/l
Aldehyde dehydrogenase 500- -50000U/l
Stabilizer 10-80% (total volume)
Reagent III
Buffer 40-200 mmol/l
Inosine monophosphate 1-50 mmol/l
Stabilizer 10-50mmol/l
The formulation of the three agents is not limited to the above formulation, and the components of the agent I, monophosphate, ethanol, oxidized coenzyme and the like, can be contained in the agent II, and the components of the agent II, nucleoside phosphatase, xanthine oxidase, catalase, aldehyde dehydrogenase and the like, can also be contained in the agent I, so that various formulations can be formed, which are not described in detail.
The pH of the buffer may range from 6.0 to 11.0. The buffer may be Tris (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) buffer, Phosphate (Phosphate) buffer, Triethanolamine (Triethanolamine) buffer, 2-Amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) buffer, Imidazole (Imidazole) buffer, Glycylglycine (glycine) buffer, or the like, but is not limited thereto.
The above oxidized coenzyme may be NADP+、NAD+Or thio-NAD+And oxidized nicotinamide coenzymes or derivatives thereof.
In addition, in order to reduce the cross influence among the components of each reagent and maintain the stability of the reagent for long-term storage, 10-80% or 10-50mmol/l of stabilizer is usually added into the above single-dose, double-dose, reagent I, reagent II or triple-dose reagent I, reagent II or reagent III, and the stabilizer can be one or more of ethylene glycol, propylene glycol, glycerol, glycan, polyalcohol, ammonium sulfate or salt.
Experiments show that the 5' -nucleotidase diagnostic kit is more ideal in the following formula component relationship no matter single, double or triple dose in consideration of the accuracy of the determination result and the economy of the preparation cost:
buffer solution 80-120 mmol/l
Inosine monophosphate 1-5 mmol/l
Monophosphate 2-10 mmol/l
Nucleoside phosphatase 5000-10000U/l
Xanthine oxidase 5000-10000U/l
Ethanol 3-12 mmol/l
Catalase 5000-10000U/l
Oxidized coenzyme 1-5 mmol/l
Aldehyde dehydrogenase 5000-10000U/l
Stabilizer 10-80% (total volume)
The invention fully utilizes the enzymology method, and the enzymolysis reaction has the characteristic of high specificity and is not easily interfered by other substances such as internal sources and external sources. The enzymatic method is simple and convenient and is easy to operate. The specificity of the enzymatic reaction promotes accurate test results. The enzymolysis reaction is carried out under the condition of buffer solution, and the environmental pollution problem is avoided. The enzyme method does not need special and extra instruments, and has low test cost. Therefore, the method can ensure higher test accuracy and is more convenient to popularize and apply.
In addition, one of the remarkable features of the present invention is that it can eliminate contamination of internal and external sources of inosine and hypoxanthine, the effect of eliminating the internal and external sources of inosine and hypoxanthine occurring in the first half of the entire reaction period, the contaminated internal and external sources of inosine and hypoxanthine having been consumed in the latter half, and inosine and hypoxanthine required for testing the activity of 5 '-nucleotidase in the latter half are both produced from the activity of 5' -nucleotidase.
Detailed Description
The present invention will be further described with reference to the following examples.
EXAMPLE one (Single dose)
The 5' -nucleotidase diagnostic kit of the present embodiment includes:
buffer 80mmol/l
Inosine monophosphate 1mmol/l
Monophosphate 2mmol/l
Nucleoside phosphatase 5000U/l
Xanthine oxidase 5000U/l
Ethanol 3mmol/l
Catalase 5000U/l
Oxidized coenzyme 1mmol/l
Aldehyde dehydrogenase 5000U/l
50% stabilizer (total volume)
Setting on a full-automatic biochemical analyzer: the temperature is 37 ℃, the reaction time is 10 minutes, the test main wavelength is 340nm, the test auxiliary wavelength is more than 405nm, the volume ratio of the tested 5' -nucleotidase sample to the reagent is 1/25, the reaction direction is positive reaction, the delay time is 1 minute, the detection time is 2 minutes, and the theoretical K value is-4180.
After the sample and reagents are added, they are mixed and thefollowing reaction occurs:
inosine monophosphate + water5' -nucleotidaseInosine + monophosphate
Inosine + monophosphateNucleoside phosphatasesHypoxanthine + 1-ribose phosphate
Hypoxanthine + water + oxygenXanthine oxidaseUrate + hydrogen peroxide
Hydrogen peroxide + ethanolHydrogen peroxideEnzymeAcetaldehyde + water
And (3) placing the final reactant under a biochemical analyzer, and detecting the ascending speed of the absorbance with the main wavelength of 340nm so as to measure and calculate the activity of the 5' -nucleotidase.
In this example, the enzyme coupling reaction system such as 5 '-nucleotidase, nucleoside phosphatase, xanthine oxidase, catalase, aldehyde dehydrogenase, etc. was used to finally reduce the oxidized coenzyme to the reduced coenzyme, since the reduced coenzyme has an absorption peak at 340nm, the increase of the absorption peak at 340nm can directly reflect the activity of 5' -nucleotidase.
EXAMPLE two (two-agent)
The 5' -nucleotidase diagnostic reagents of the present example were:
reagent I
Buffer 100mmol/l
Monophosphate 6mmol/l
8000U/l nucleoside phosphatase
Xanthine oxidase 8000U/l
Ethanol 8mmol/l
Catalase 8000U/l
Oxidized coenzyme 3mmol/l
Aldehyde dehydrogenase 8000U/l
50% stabilizer (total volume)
Reagent II
Buffer 100mmol/l
Inosine monophosphate 3mmol/l
Stabilizer 30mmol/l
When the activity of the 5 '-nucleotidase is measured, the temperature is controlled at 30 ℃, the reaction time is 15 minutes, the main test wavelength is 340nm, the sub-test wavelength is more than 405nm, the volume ratio of the 5' -nucleotidase sample to be measured to the reagent is 1/25, the reaction direction is positive reaction, the delay time is 1 minute, the detection time is 2 minutes, and the theoretical K value is-4180.
The specific determination steps are as follows:
inosine monophosphate + water5' -nucleotidaseInosine + monophosphate
Inosine + monophosphateNucleoside phosphatasesHypoxanthine + 1-ribose phosphate
Hypoxanthine + water + oxygenXanthine oxidaseUrate + hydrogen peroxide
Hydrogen peroxide + ethanolCatalase enzymeAcetaldehyde + water
Acetaldehyde, oxidized coenzyme and waterAldehyde dehydrogenaseAcetic acid + reducing coenzyme + hydrogen ion
And (3) placing the final reactant under a biochemical analyzer, and detecting the ascending speed of the absorbance with the main wavelength of 340nm so as to measure and calculate the activity of the 5' -nucleotidase.
The reaction time of each reaction step was controlled to 15 minutes.
EXAMPLE three (three doses)
The 5' -nucleotidase diagnostic reagent of the present example was three agents, including:
reagent I
Buffer 120mmol/l
Monophosphate 10mmol/l
Ethanol 12mmol/l
Oxidized coenzyme 5mmol/l
50mmol/l stabilizer
Reagent II
Buffer 120mmol/l
10000U/l nucleoside phosphatase
Xanthine oxidase 10000U/l
Catalase 10000U/l
Aldehyde dehydrogenase 10000U/l
50% stabilizer (total volume)
Reagent III
Buffer 120mmol/l
Inosine monophosphate 5mmol/l
50mmol/l stabilizer
Setting on a full-automatic biochemical analyzer: the temperature is 25 ℃, the reaction time is 20 minutes, the test main wavelength is 340nm, the test auxiliary wavelength is more than 405nm, the volume ratio of the tested 5' -nucleotidase sample to the reagent is 1/25, the reaction direction is positive reaction, the delay time is 1 minute, the detection time is 2 minutes, and the theoretical K value is-4180.
The specific determination steps are as follows:
inosine monophosphate + water5' -nucleotidaseInosine + monophosphate
Inosine + monophosphateNucleoside phosphatasesHypoxanthine + 1-ribose phosphate
Hypoxanthine + water + oxygenXanthine oxidaseUrate + hydrogen peroxide
Hydrogen peroxide + ethanolCatalase enzymeAcetaldehyde + water
Acetaldehyde, oxidized coenzyme and waterAldehyde dehydrogenaseAcetic acid + reductionProtogenic coenzyme + hydrogen ion
And (3) placing the final reactant under a biochemical analyzer, and detecting the ascending speed of the absorbance with the main wavelength of 340nm so as to measure and calculate the activity of the 5' -nucleotidase.
The reaction time of each reaction step was controlled to 20 minutes.
Example four
The 5' -nucleotidase diagnostic reagents of the present example were:
reagent I
Buffer 100mmol/l
Monophosphate 5mmol/l
12000U/l nucleoside phosphatase
Xanthine oxidase 12000U/l
Ethanol 8mmol/l
Catalase 12000U/l
Oxidized coenzyme 3mmol/l
Aldehyde dehydrogenase 12000U/l
50% stabilizer (total volume)
Reagent II
Buffer 100mmol/l
Adenosine monophosphate 3mmol/l
40mmol/l stabilizer
Setting on a biochemical analyzer: the temperature is 37 ℃, the reaction time is 10 minutes, the test main wavelength is 340nm, the test auxiliary wavelength is more than 405nm, the volume ratio of the tested 5' -nucleotidase sample to the reagent is 1/25, the reaction direction is positive reaction, the delay time is 1 minute, the detection time is 2 minutes, and the theoretical K value is-4180.
After the sample and reagents are added, they are mixed and the following reaction occurs:
inosine monophosphate + water5' -nucleotidaseInosine + monophosphate
Inosine + monophosphateNucleoside phosphatasesHypoxanthine + 1-ribose phosphate
Hypoxanthine + water + oxygenXanthine oxidaseUrate + hydrogen peroxide
Hydrogen peroxide + ethanolCatalase enzymeAcetaldehyde + water
Acetaldehyde, oxidized coenzyme and waterAldehyde dehydrogenaseAcetic acid + reducing coenzyme + hydrogen ion
And (3) placing the final reactant under a biochemical analyzer, and detecting the ascending speed of the absorbance with the main wavelength of 340nm so as to measure and calculate the activity of the 5' -nucleotidase.
In a word, experiments prove that the measuring method can obtain the required measuring result completely by an ultraviolet/visible light analyzer or a semi-automatic or full-automatic biochemical analyzer, has high sensitivity and good precision, and is not polluted by internal and external substances.
Claims (9)
1. A method for measuring the activity of 5' -nucleotidase comprises the following steps:
1) mixing the sample with a reagent mainly consisting of inosine monophosphate, nucleoside phosphatase, xanthine oxidase, ethanol, catalase, oxidized coenzyme and aldehyde dehydrogenase, and reacting the following reaction:
inosine monophosphate + water5' -nucleotidaseInosine + monophosphate
Inosine + monophosphateNucleoside phosphatasesHypoxanthine + 1-ribose phosphate
Hypoxanthine + water + oxygenXanthine oxidaseUrate + hydrogen peroxide
Hydrogen peroxide + ethanolCatalase enzymeAcetaldehyde + water
Acetaldehyde, oxidized coenzymeand waterAldehyde dehydrogenaseAcetic acid + reducing coenzyme + hydrogen ion
2) And detecting the descending speed of the final reactant at the main wavelength of 340nm to measure and calculate the activity of the 5' -nucleotidase.
2. The method for measuring 5' -nucleotidase activity according to claim 1, wherein: and step 2) is to place the final reactant under an ultraviolet/visible light analyzer or a semi-automatic or full-automatic biochemical analyzer, detect the dominant wavelength of 340nm and the descending speed of absorbance, and measure and calculate the activity of the 5' -nucleotidase.
3. The method for measuring 5' -nucleotidase activity according to claim 1 or 2, wherein: the temperature is controlled within the range of 20 ℃ to 50 ℃, and the reaction time is controlled within 2-30 minutes.
4. The method for measuring 5' -nucleotidase activity according to claim 1 or 2, wherein: the ratio of the 5' -nucleotidase sample to the reagent to be detected was controlled at 1/10 to 1/250.
5. A diagnostic kit of 5' -nucleotidase comprises the following components:
buffer 40-200 mmol/l
Inosine monophosphate 1-50 mmol/l
Monophosphate 0.2-10 mmol/l
Nucleoside phosphatase 500-50000U/l
Xanthine oxidase 500-50000U/l
Ethanol 1-30 mmol/l
Catalase 500- -50000U/l
Oxidized coenzyme 0.5-20 mmol/l
Aldehyde dehydrogenase 500- -50000U/l
Stabilizer 10-80% (total volume)
6. The diagnostic kit for 5' -nucleotidase according to claim 5, wherein: the reagent is prepared into single, double or triple dose.
7. The diagnostic kit for 5' -nucleotidase according to claim 5 or 6, wherein: the stabilizer is at least one of ethylene glycol, propylene glycol, glycerol, glycan, polyalcohol, ammonium sulfate or salt.
8. The diagnostic kit for 5' -nucleotidase according to claim 5 or 6, wherein: the buffer has a pH in the range of 6.0 to 11.0, and is one of a tris (carboxymethyl) aminomethane-hydrochloric acid buffer, a phosphate buffer, a triethanolamine buffer, a 2-amino-2-methyl-1-propanol buffer, an imidazole buffer, or a glycylglycine buffer.
9. The diagnostic kit for 5' -nucleotidase according to claim 5 or 6, wherein: the oxidized coenzyme is NADP+、NAD+Or thio-NAD+Oxidized nicotinamide coenzyme or one of its derivatives.
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| CN 200410064906 CN1760369A (en) | 2004-10-11 | 2004-10-11 | Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase |
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| CN 200410064906 CN1760369A (en) | 2004-10-11 | 2004-10-11 | Method for testing activity of 5'-nucleotidase, and diagnosis kit for 5'-nucleotidase |
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