CN101173901A - N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase - Google Patents
N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase Download PDFInfo
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- CN101173901A CN101173901A CNA2006100973336A CN200610097333A CN101173901A CN 101173901 A CN101173901 A CN 101173901A CN A2006100973336 A CNA2006100973336 A CN A2006100973336A CN 200610097333 A CN200610097333 A CN 200610097333A CN 101173901 A CN101173901 A CN 101173901A
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Abstract
The invention relates to an N-acetyl-beta-glucosaminidase diagnostic reagent kit and a method for testing the activity of N-acetyl-beta-glucosaminidase as well as composition and content of the reagent, belonging to the technical field of medical testing. The invention is characterized in that composition in the diagnostic reagent kit comprises buffer solution, m-Cresolsulfonphthaleinyl-beta-acetamido-D-glucoside and stabilizing agent; the sample is mixed with the reagent according to specified ratio to generate a series enzymatic reactions, then the reactant is arranged under a ultraviolet/visible light analyzer to test the increase speed of absorbance at wavelength of 580nm, thereby the activity of N-acetyl-beta-glucosaminidase can be tested. The invention has the advantages that the test results can be easily obtained using ultraviolet/visible light analyzer; therefore, the popularization and application are facilitated.
Description
Technical field
The present invention relates to a kind of N-acet-beta-amino glucosidase (EC 3.2.1.30) diagnostic kit, the invention still further relates to simultaneously and measure the active method of N-acet-beta-amino glucosidase (EC 3.2.1.30), belong to medical test determination techniques field.
Background technology
N-acetyl-(NAG) is lysosomal a kind of acid hydrolase in the cell.Result of study shows that the size of NAG vigor can be used as effective auxiliary diagnosis index of multiple disease in people's body fluid (blood, urine), and the NAG vigor has important clinic value in the detection body fluid.NAG can be used as very sensitive index of injury of kidney, is caused in the acute renal failure patient urine by shock and extremely increases, and reaches as high as 1200 times of normal person.Acute glomerulonephritis, nephrotic syndrome, poisonous substance or the reaction of drug-induced renal toxicity all can make NAG increase.After the kidney transplant, enzymatic determination can be used to judge and has or not rejection.Pathologic increases and sees various parenchymal lesion of the kidney, the kidney transplant rejection, and the renal toxicity drug use is too much.Some glomerulonephritis, nephrotic syndrome also have part to raise.
The mensuration of NAG vigor mainly contains fluorescence spectrophotometry and visible spectrophotometry.The former costs an arm and a leg because of required instrument, and should not be used as the routine inspection of general hospital to having relatively high expectations of substrate solution preparation; Though the reported method of the latter is each has something to recommend him selecting of various experiment conditions, the troublesome poeration that has is time-consuming, and the selected conditioned disjunction reagent stability that has is not good enough, is difficult to satisfy the clinical examination requirement.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) technology of utilizing, continuous monitoring is in the variation of 580nm wavelength place absorbance, measured the method for N-acetyl-beta-amino glucosaccharase activity, simultaneously, the present invention also will provide in order to realize the N-acet-beta-amino glucosidase diagnostic reagent box of this method, adopt this kit not only can be visible light analysis instrument or half, carrying out the N-acetyl-beta-amino glucosaccharase activity on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
N-acetyl-beta-amino glucosaccharase activity assay method principle of the present invention is as follows:
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides
Acetyl glucosaminidase
Metacresol sulphonyl dioctyl phthalate strategic point Yin+acetylglucosamine glycosides
This method is used N-acet-beta-amino glucosidase enzymatic reaction continuous monitoring method.N-acet-beta-amino glucosidase enzymolysis metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides reaction produces metacresol sulphonyl dioctyl phthalate strategic point Yin base (has absorption peak at the 580nm place, molar extinction coefficient is 40.670), by measuring the speed that 580nm place absorbance rises, can calculate the active size of N-acet-beta-amino glucosidase.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, and no matter be single agent or two agent, the N-acet-beta-amino glucosidase diagnostic reagent box of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides 2mmol/L
N-acet-beta-amino glucosidase diagnostic reagent box of the present invention can be single agent, comprising: damping fluid, stabilizing agent, metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent.
Reagent 2
Damping fluid, stabilizing agent, metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides.
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-can not limit by the position of acetylglucosamine glycosides in reagent 1 or reagent 2.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The N-acet-beta-amino glucosidase diagnostic reagent of present embodiment is a single reagent, comprising:
Imidazoles/hydrochloride buffer 50mmol/L
Stabilizing agent 50mmol/L
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides 2mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 580nm, test commplementary wave length 660nm, the volume ratio of tested N-acet-beta-amino glucosidase sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value 639.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 580nm absorbance rises, thereby calculates the active size of N-acet-beta-amino glucosidase.
Embodiment two
The N-acet-beta-amino glucosidase diagnostic reagent of present embodiment is a double reagent, comprising:
Reagent 1
Sodium hydrogen phosphate-citrate buffer solution 50mmol/L
Stabilizing agent 50mmol/L
Reagent 2
Sodium hydrogen phosphate-citrate buffer solution 50mmol/L
Stabilizing agent 50mmol/L
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides 2mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 580nm, test commplementary wave length 660nm, the volume ratio of tested N-acet-beta-amino glucose battalion's enzyme sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), about about 1 minute of time delay, about 2 minutes detection times, theoretical k value 332.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the speed that predominant wavelength 580nm absorbance rises, thereby calculates the active size of N-acet-beta-amino glucosidase.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, be not subjected in the pollution of allogenic material, easy to utilize.
Claims (5)
1. N-acetyl-beta-amino glucosaccharase activity assay method, its method principle is as follows:
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides
Acetyl glucosaminidase
Metacresol sulphonyl dioctyl phthalate strategic point Yin+acetylglucosamine glycosides
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides
m-Cresolsulfonphthaleinyl-N-acetyl-D-glucosamine
Metacresol sulphonyl dioctyl phthalate strategic point Yin
m-Cresolsulfonphthalein
The end reaction thing is placed under visible light analysis instrument or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 580nm absorbance rises, calculate the active size measurement result of N-acet-beta-amino glucosidase.
2. N-acet-beta-amino glucosidase diagnostic reagent box, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-50mmol/L
Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides
0.2-10mmol/L
It is characterized in that: kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
3. according to the described N-acet-beta-amino glucosidase diagnostic reagent of claim 2 box, it is characterized in that: form single agent reagent by damping fluid, stabilizing agent, metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides.
4. according to the described N-acet-beta-amino glucosidase diagnostic reagent of claim 2 box, it is characterized in that: form two agent reagent by damping fluid, stabilizing agent, metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides; Reagent 1 is made up of damping fluid, stabilizing agent; Reagent 2 is made up of damping fluid, stabilizing agent, metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-acetylglucosamine glycosides.Metacresol sulphonyl dioctyl phthalate strategic point Yin base-β-can not limit by the position of acetylglucosamine glycosides in reagent 1 or reagent 2.
5. according to the described N-acet-beta-amino glucosidase diagnostic reagent of claim 2 box, it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100973336A CN101173901A (en) | 2006-10-30 | 2006-10-30 | N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100973336A CN101173901A (en) | 2006-10-30 | 2006-10-30 | N-acet-beta-amino glucosidase diagnostic reagent kit and method for measuring active concentration of N-acet-beta-amino glucosidase |
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| CN101173901A true CN101173901A (en) | 2008-05-07 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102128795A (en) * | 2010-11-19 | 2011-07-20 | 浙江大学 | Method for testing freshness of propolis |
| CN101738379B (en) * | 2009-12-31 | 2012-04-25 | 宁波美康生物科技股份有限公司 | Liquid reagent for determining 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 |
| CN106442503A (en) * | 2016-09-30 | 2017-02-22 | 广州鸿琪光学仪器科技有限公司 | Acetylglucosaminidasedetection reagent, reaction pad, preparation method of reaction pad and kit |
-
2006
- 2006-10-30 CN CNA2006100973336A patent/CN101173901A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101738379B (en) * | 2009-12-31 | 2012-04-25 | 宁波美康生物科技股份有限公司 | Liquid reagent for determining N-acetyl-beta-D-glucosaminidase |
| CN102128795A (en) * | 2010-11-19 | 2011-07-20 | 浙江大学 | Method for testing freshness of propolis |
| 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 |
| CN106442503A (en) * | 2016-09-30 | 2017-02-22 | 广州鸿琪光学仪器科技有限公司 | Acetylglucosaminidasedetection reagent, reaction pad, preparation method of reaction pad and kit |
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