CN1995975A - Method for detecting N-acetyl-beta-amino glucosaccharase activity and diagnosis kit therefor - Google Patents
Method for detecting N-acetyl-beta-amino glucosaccharase activity and diagnosis kit therefor Download PDFInfo
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- CN1995975A CN1995975A CN 200610161229 CN200610161229A CN1995975A CN 1995975 A CN1995975 A CN 1995975A CN 200610161229 CN200610161229 CN 200610161229 CN 200610161229 A CN200610161229 A CN 200610161229A CN 1995975 A CN1995975 A CN 1995975A
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- acetyl
- glucosaminidase
- nitrobenzene
- acetylglucosaminide
- activity
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Abstract
The active feature inspection for acetylglucosamidase and its diagnostic agent box uses its fostering of continuous monitoring method, enzymolysising dual chlorineor chlorine acetylglucosamidase reaction, through the absorbance elevating speed of the 405nm, inspecting its active feature. The agent box comprises the bumper solution, synthesized nitrobenzene betaacetylglucosamidase and stabilizer to make dual agent by the diagnostic agent box to reduce cross impact of all kinds of components. Through visible light analyzer, the result is precise, free from outside contamination, easy for promotion.
Description
Technical Field
The invention relates to detection of N-acetyl- β -glucosaminidase activity in the field of medicine, in particular to a method for determining the activity of N-acetyl- β -glucosaminidase by using an enzymatic colorimetric method, and an N-acetyl- β -glucosaminidase diagnostic kit prepared by the method, belonging to the technical field of analysis and detection of the activity of N-acetyl- β -glucosaminidase.
Background
The research shows that the activity of NAG in human body fluid (blood and urine) can be used as an effective auxiliary diagnosis index of various diseases, and the detection of the activity of NAG in the body fluid has important clinical value.
The NAG activity is mainly measured by fluorescence spectrophotometry and visible spectrophotometry. The former is not suitable for routine examination in general hospitals because the needed instruments are expensive and the requirement on the preparation of substrate solution is high; the latter reported method is different in the selection of various experimental conditions, but some methods are complicated and time-consuming in operation, and some selected conditions or reagents are poor in stability and difficult to meet the requirements of clinical examination.
Disclosure of Invention
The invention aims to provide a method for measuring the activity of N-acetyl- β -glucosaminidase and an N-acetyl- β -glucosaminidase diagnostic kit prepared by applying the method.
In order to achieve the purpose of the invention, the method for measuring the activity of the N-acetyl- β -glucosaminidase by using the enzymatic colorimetric method comprises the following steps:
firstly, a sample to be tested is evenly mixed with a reagent containing synthetic nitrobenzene- β -acetylglucosaminide to cause the following reaction,
then, the final reactant is placed under a visible light analyzer or a semi-automatic or full-automatic biochemical analyzer, the velocity of the increase in absorbance at 405nm of the dominant wavelength is detected, and the activity of the N-acetyl- β -glucosaminidase is measured.
In the method for measuring the activity of the N-acetyl- β -glucosaminidase, the synthesized nitrobenzene- β -glucosaminidase substrate is 3, 4-dinitrobenzene- β -glucosaminidase or 2-chloro-4-nitrobenzene- β -glucosaminidase, and the stabilizer is at least one of ammonium sulfate, glycerol, propylene glycol and ethylene glycol.
The N-acetyl- β -glucosaminidase diagnostic kit can be a single agent and comprises the following components:
20 to 500mmol/L of a buffer solution,
5 to 100g/ml of a stabilizer,
and (3) synthesizing nitrobenzene- β -acetylglucosaminide by 0.2-10 mmol/L.
The kit can be in a dry powder state, and is used after being dissolved by adding water before use; or can be prepared into liquid reagent for direct use.
The above components in single dose can also be combined to prepare double dose, such as:
the formula of the double agent is not limited to the formula listed in the table, wherein the synthesized nitrobenzene- β -acetylglucosamine in the agent II can be put into the agent I, so that various formulas can be formed, and the formula is not listed.
The method for determining the activity of the N-acetyl- β -glucosaminidase comprises a single agent or a double agent, wherein the synthesized nitrobenzene- β -glucosaminidase substrate is 3, 4-dinitrobenzene- β -glucosaminidase or 2-chloro-4-nitrobenzene- β -glucosaminidase.
In addition, in order to reduce the cross effect among the components of the reagents and maintain the stability of the reagents for long-term storage, a stabilizer is usually added into the single-dose or double-dose reagent I/reagent II, and the concentration is within the range of 0.1-3 mol/L, or 5-100 g/ml, or 5-50% v/v.
Substances used as stabilizers may be: at least one of ammonium Sulfate (ammonium Sulfate), Glycerol (Glycerol), Propylene Glycol (Propylene Glycol), and Ethylene Glycol (Ethylene Glycol).
Research shows that the diagnostic kit is relatively ideal and also a preferred scheme of the invention in terms of the following formula component relationship no matter single-dose or double-dose due to the comprehensive consideration of the accuracy of the determination result and the economy of the preparation cost:
the concentration of the buffer solution is 100mmol/L,
2mol/L of a stabilizing agent is added,
2mmol/L nitrobenzene- β -acetylglucosaminide was synthesized.
The invention utilizes the technology of enzyme Colorimetric Method, continuously monitors the change of absorbance at 405nm wavelength, and obtains the Method for measuring the activity of N-acetyl- β -glucosaminidase, and simultaneously, the invention also provides a diagnostic kit of N-acetyl- β -glucosaminidase for realizing the Method, and the diagnostic kit can be used for measuring the activity of N-acetyl- β -glucosaminidase on a visible light analyzer or a semi-automatic or full-automatic biochemical analyzer, and has high measuring speed and high accuracy, thereby being practically popularized and applied.
The outstanding substantive features and remarkable progress of the technical scheme of the invention are mainly shown in the following steps:
(1) the method utilizes the enzymatic colorimetric method technology to determine the activity of the N-acetyl- β -glucosaminidase, and the test result is accurate;
(2) the components participating in the reaction are added externally, so that the test device is not polluted by internal and external substances, and the test process has high accuracy;
(3) the method is simple and easy to operate, can quickly obtain a detection result, and the reaction is carried out under the condition of a buffer solution, so that the environment is not polluted;
(4) the method can be used for rapid detection on a common ultraviolet/visible light analyzer or a semi-automatic/full-automatic biochemical analyzer, does not need special or additional instruments, has low test cost and is convenient for popularization and application in the industry;
(5) the determination method provided by the invention can be used for preparing reagents in various forms such as liquid reagents, dry powder reagents and the like, and is used for determining the activity of the N-acetyl- β -glucosaminidase in various samples;
(6) the liquid N-acetyl- β -glucosaminidase diagnostic kit provided by the invention has good stability, well ensures the application test effect, is prepared into double agents, can further reduce the cross influence among various components, has more credible detection result and more stable agent, and can be stored for a long time.
Detailed Description
The invention relates to a method for determining the activity of N-acetyl- β -glucosaminidase and a diagnostic kit of N-acetyl- β -glucosaminidase, which applies a continuous monitoring method of N-acetyl- β -glucosaminidase enzymatic reaction, and carries out enzymatic hydrolysis on 3, 4-dinitrobenzene- β -glucosaminidase or 2-chloro-4-nitrobenzene- β -glucosaminidase to generate 3, 4-dinitrobenzene or 2-chloro-4-nitrobenzene (an absorption peak exists at 405nm, the molar extinction coefficient is 18.45) by the N-acetyl- β -glucosaminidase, and calculates the activity of the N-acetyl- β -glucosaminidase by measuring the rising speed of absorbance at 405 nm.
The technical solution of the present invention is further illustrated by the following specific examples. These examples are merely exemplary applications and are not to be construed as limiting the scope of the invention as claimed.
EXAMPLE one (Single dose)
The N-acetyl- β -glucosaminidase diagnostic kit is prepared from the following components in parts by weight:
50mmol/L of imidazole/hydrochloric acid buffer solution,
the ammonium sulfate is 80g/ml, and the concentration of the ammonium sulfate is 80g/ml,
2mmol/L of 3, 4-dinitrobenzene- β -acetamido glucoside.
After the reagents are completely dissolved and prepared, subpackaging the mixture into bottles, and carrying out freeze drying to prepare dry powder reagents; before use,purified water is added for re-dissolving.
The temperature is 37 ℃, the reaction time is 10 minutes, the initial absorbance is less than or equal to 0.1, the test main wavelength is 405nm, the test auxiliary wavelength is 505nm, the volume ratio of the tested N-acetyl- β -glucosaminidase sample to the reagent is 1: 25, the reaction direction is positive reaction (rising reaction), the delay time is about 1 minute, the detection time is about 2 minutes, and the theoretical K value is 1409.
After the sample and the reagent are added, the mixture is mixed and reacts, and finally the reactant is placed under a biochemical analyzer to detect the ascending speed of the absorbance with the main wavelength of 405nm, so that the activity of the N-acetyl- β -glucosaminidase is measured and calculated.
EXAMPLE two (two-agent)
The N-acetyl- β -glucosaminidase diagnostic kit is prepared from the following components in parts by weight:
reagent I-
50mmol/L of disodium hydrogen phosphate-citric acid buffer solution,
2mol/L of glycerol;
reagent II- (III) -
50mmol/L of disodium hydrogen phosphate-citric acid buffer solution,
propylene glycol 50% v/v of propylene glycol,
2-chloro-4-nitrobenzene- β -acetamido glucoside 2 mmol/L.
And after the reagents are completely dissolved and prepared, subpackaging the mixture into bottles to prepare the liquid double reagent which can be directly used.
Setting the temperature to 37 ℃ on a full-automatic biochemical analyzer, the reaction time to 10 minutes, the initial absorbance to be less than or equal to 0.1, the test main wavelength to be 405nm, the test auxiliary wavelength to be 505nm, the volume ratio of the tested N-acetyl- β -glucosaminidase sample to the reagent I and the reagent II to be 2: 20: 5, the reaction direction to be positive and negative reaction (rising reaction), the delay time to be about 1 minute, the detection time to be about 2 minutes and the theoretical K value to be 732.
After the sample and the reagent are added, the mixture is mixed and reacts, and finally the reactant is placed under a biochemical analyzer to detect the ascending speed of the absorbance with the main wavelength of 405nm, so that the activity of the N-acetyl- β -glucosaminidase is measured and calculated.
In a word, experiments prove that the measuring method can obtain the required measuring result completely by a common biochemical analyzer, has high sensitivity and good precision, is not polluted by internal and external substances, and is convenient to popularize and apply.
Claims (7)
- A method for measuring the activity of N-acetyl- β -glucosaminidase, comprising the steps of:① mixing the sample to be tested with the reagent containing synthetic nitrobenzene- β -acetyl glucosaminide uniformly to make it react,② placing the final reactant under a visible light analyzer or a semi-automatic or full-automatic biochemical analyzer, detecting the ascending speed of the absorbance of the main wave length at 405nm, and calculating the activity of the N-acetyl- β -glucosaminidase.
- 2. The method of determining the activity of N-acetyl- β -aminoglycoside according to claim 1, wherein the substrate for the synthesis of nitrobenzene- β -acetylglucosaminide is 3, 4-dinitrobenzene- β -acetylglucosaminide or 2-chloro-4-nitrobenzene- β -acetylglucosaminide.
- An N-acetyl- β -glucosaminidase diagnostic kit, wherein the reagent in the kit comprises the following components:20 to 500mmol/L of a buffer solution,0.1 to 3mol/L of a stabilizer, or5 to 100g/ml, or5～50％v/v，And (3) synthesizing nitrobenzene- β -acetylglucosaminide by 0.2-10 mmol/L.
- 4. The diagnostic kit of N-acetyl- β -aminoglycoside according to claim 3, wherein said synthetic nitrobenzene- β -acetylglucosaminide substrate is 3, 4-dinitrobenzene- β -acetylglucosaminide or 2-chloro-4-nitrobenzene- β -acetylglucosaminide.
- 5. The diagnostic kit of N-acetyl- β -aminoglycoside according to claim 3, wherein said stabilizer is at least one of ammonium sulfate, glycerol, propylene glycol and ethylene glycol.
- 6. The diagnostic kit for N-acetyl- β -glucosaminidase of any one of claims 3-5, characterized in that the reagent is formulated as a single agent or a double agent.
- 7. The N-acetyl- β -glucosaminidase diagnostic kit of any one of claims 3-5, characterized in that the kit is a dry powder kit or a liquid kit.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006101612299A CN100561187C (en) | 2006-12-15 | 2006-12-15 | The assay method of N-acetyl-beta-amino glucosaccharase activity and diagnostic kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006101612299A CN100561187C (en) | 2006-12-15 | 2006-12-15 | The assay method of N-acetyl-beta-amino glucosaccharase activity and diagnostic kit |
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| CN1995975A true CN1995975A (en) | 2007-07-11 |
| CN100561187C CN100561187C (en) | 2009-11-18 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277410A (en) * | 2011-08-16 | 2011-12-14 | 南京欣迪生物药业工程有限责任公司 | Quantitative detection kit for seminal plasma alpha-glycosidase activity and application thereof |
| CN103484525A (en) * | 2012-06-14 | 2014-01-01 | 北京和信非凡生物技术有限公司 | Method, substrate and reagents for alpha-N-acetylglucosaminidase activity detection |
| CN104297181A (en) * | 2014-10-10 | 2015-01-21 | 宁波大学 | Detection reagent of N-acetyl-beta-D-glucosaminidase |
| CN104297179A (en) * | 2014-10-10 | 2015-01-21 | 宁波医杰生物科技有限公司 | Reagent used for detecting N-acetyl-beta-D-glucosaminidase |
-
2006
- 2006-12-15 CN CNB2006101612299A patent/CN100561187C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277410A (en) * | 2011-08-16 | 2011-12-14 | 南京欣迪生物药业工程有限责任公司 | Quantitative detection kit for seminal plasma alpha-glycosidase activity and application thereof |
| CN103484525A (en) * | 2012-06-14 | 2014-01-01 | 北京和信非凡生物技术有限公司 | Method, substrate and reagents for alpha-N-acetylglucosaminidase activity detection |
| CN104297181A (en) * | 2014-10-10 | 2015-01-21 | 宁波大学 | Detection reagent of N-acetyl-beta-D-glucosaminidase |
| CN104297179A (en) * | 2014-10-10 | 2015-01-21 | 宁波医杰生物科技有限公司 | Reagent used for detecting N-acetyl-beta-D-glucosaminidase |
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| CN100561187C (en) | 2009-11-18 |
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