CN101173898A - Monoamine oxidase diagnostic reagent kit and method for measuring active concentration of monoamine oxidase - Google Patents
Monoamine oxidase diagnostic reagent kit and method for measuring active concentration of monoamine oxidase Download PDFInfo
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- CN101173898A CN101173898A CNA2006100973251A CN200610097325A CN101173898A CN 101173898 A CN101173898 A CN 101173898A CN A2006100973251 A CNA2006100973251 A CN A2006100973251A CN 200610097325 A CN200610097325 A CN 200610097325A CN 101173898 A CN101173898 A CN 101173898A
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Abstract
The invention relates to a monoamine oxidase diagnosis reagent kit which adopts the techniques of enzymatic colorimetric method and enzyme couple reaction, belonging to the technical field of medical test and determination. The invention also relates to the method principle of determining the active concentration of monoamine oxidase, and the reagent composition and components. The component of the reagent kit mainly comprises buffer solution, reduced coenzyme, amine compounds, NAD (P) H oxidase and stabilizer. The method for determining the active concentration of monoamine oxidase is as follow: mixing the sample and the reagent according to a certain volume ratio to perform a series of enzymatic reactions; placing the reactant under an ultraviolet / visible light analyzer to detect the ascending speed of the absorbency at the main wavelength of 340 nm; finally determining the active concentration of monoamine oxidase. The invention has the advantages that the required determining result is obtained only with the ultraviolet / visible light analyzer.
Description
Technical field
The present invention relates to a kind of monoamine oxidase diagnosing reagent kit, the invention still further relates to the method for measuring monoamine oxidase activity concentration simultaneously, belong to medical test determination techniques field.
Background technology
Monoamine oxidase (MAO) is measured the enzyme process that can be divided into chemical spectrophotometric method, fluorescence method, immunodepression and apply for a patent now.Generally use chemical spectrophotometric method, the monoamine oxidase reaction substrate also can be used the H that monoamine oxidase catalysis amine discharges with benzylamine (Benzylamine) and P-Toluidine-β-azo naphthols more
2O
2The oxidation colour former, as 10-N-methylamino formoxyl-3,7-dimethylamino-10-hydrogen-phenothiazine (MCDP).In addition, the monoamine oxidase reaction substrate also has butylamine (butylamine), amylamine (amylamine), β-phenethyl amine (b-phenyl ethylamine), tyrasamine (tyramine:3-Uteramin 3-parahdroxyphenylethylamine), serotonin (5-hydroxytryptamine) etc., butylamine, amylamine, β-phenethyl amine are about 30% of 3-Uteramin activity, use benzene methanamine usually as the activity of monoamine oxidase height of substrate than other substrates.At present to measure many be substrate with benzylamine and P-Toluidine-β-azo naphthols to monoamine oxidase, but P-Toluidine-β-azonaphthalene Phenoxybenzamine method need extract with cyclohexane, limited the practicality of this method, and can not use the full automatic biochemical apparatus analysis; The benzylamine ratio juris is that benzylamine generates benzyl aldehyde under the effect of monoamine oxidase, then under the condition of strong basicity NaOH with dinitrophenylhydrazine reaction, generate the aldehyde phenylhydrazone, on biochemical instruments, measure also difficulty.
Fluorescence method detect monoamine oxidase be with β-phenyl ethylamine as substrate, it is the Km maximum when 10 ~ 100mg, is higher than the Km value of benzylamine and serotonin.
Immunological method is to utilize monoamine oxidase antibody and monoamine oxidase to react, and carries out the monoamine oxidase isodynamic enzyme after the effect and measures.Using more monoclonal antibody at present is MAO-A3C9, MAO-A4F10, MAO-A7B10, MAO-A7E10 and MAO-B1C2 etc.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for monoamine oxidase activity concentration, simultaneously, the present invention also will provide in order to realize the monoamine oxidase diagnosing reagent kit of this method, adopt this kit not only can be ultraviolet analyser or half, carry out determining monoamine oxidase activity concentration on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Method for determining monoamine oxidase activity concentration principle of the present invention is as follows:
This method is used monoamine oxidase (idol) connection NAD (P) H oxidase enzymatic reaction continuous monitoring method.The reaction of monoamine oxidase enzymolysis aminated compounds produces hydrogen peroxide, again by the oxidasic effect of (idol) associating NAD (P) H, oxidized coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured the speed that reduced coenzyme rises in 340nm place absorbance, by measuring the speed that 340nm place absorbance rises, can calculate the active concentration size of monoamine oxidase.
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 monoamine oxidase diagnosing reagent kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 6mmol/L
Aminated compounds 8mmol/L
NAD (P) H oxidase 9000U/L
Monoamine oxidase diagnosing reagent kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, aminated compounds, NAD (P) H oxidase.
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, coenzyme, aminated compounds.
Reagent 2
Damping fluid, stabilizing agent, NAD (P) H oxidase.
Coenzyme, aminated compounds, the position of NAD (P) H oxidase in reagent 1 or reagent 2 can not limit.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 three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme.
Reagent 2
Damping fluid, stabilizing agent, aminated compounds.
Reagent 3
Damping fluid, stabilizing agent, NAD (P) H oxidase.
Coenzyme, aminated compounds, the position of NAD (P) H oxidase in reagent 1, reagent 2 or reagent 3 can not limit.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.
No matter be single agent, two agent or three doses, the present invention measures the method for monoamine oxidase activity concentration, and its coenzyme can be NADP
+, NAD
+Or thio-NAD
+In a kind of.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The monoamine oxidase diagnosing reagent of present embodiment is a single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 6mmol/L
Aminated compounds 8mmol/L
NAD (P) H oxidase 9000U/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≤0.05, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested monoamine oxidase 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-4180.
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 340nm absorbance rises, thereby calculates the active concentration size of monoamine oxidase.
Embodiment two
The monoamine oxidase diagnosing reagent of present embodiment is a double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 8mmol/L
Aminated compounds 12mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
NAD (P) H oxidase 12000U/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≤0.05, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested monoamine oxidase 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-2170.
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 340nm absorbance rises, thereby calculates the active concentration size of monoamine oxidase.
Embodiment three
The monoamine oxidase diagnosing reagent of present embodiment is three reagent, comprising:
Reagent 1
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Reagent 2
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Aminated compounds 6mmol/L
Reagent 3
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
NAD (P) H oxidase 6000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring monoamine oxidase activity concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.05, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested monoamine oxidase sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, and 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-2170.
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 340nm absorbance rises, thereby calculates the active concentration size of monoamine oxidase.
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 (6)
1. method for determining monoamine oxidase activity concentration, its method principle is as follows:
Aminated compounds+water+oxygen
Monoamine oxidaseCorresponding aldehyde product+ammonium ion
+ hydrogen peroxide
Hydrogen peroxide+coenzyme
NAD (P) H oxidaseReduced coenzyme+oxygen
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance rises, calculate the active concentration size measurement result of monoamine oxidase.
2. monoamine oxidase diagnosing reagent kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-50mmol/L
DPN diphosphopyridine nucleotide-10mmol/L
Aminated compounds 1-30mmol/L
NAD (P) H oxidase 1000-50000U/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 monoamine oxidase diagnosing reagent kit of claim 2, it is characterized in that: form single agent reagent by damping fluid, stabilizing agent, coenzyme, aminated compounds, NAD (P) H oxidase (EC 1.6.3.1).
4. according to the described monoamine oxidase diagnosing reagent kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, aminated compounds, NAD (P) H oxidase (EC 1.6.3.1); Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, aminated compounds; Reagent 2 is made up of damping fluid, stabilizing agent, NAD (P) H oxidase (EC 1.6.3.1).Coenzyme, aminated compounds, the position of NAD (P) H oxidase in reagent 1 or reagent 2 can not limit.
5. according to the described monoamine oxidase diagnosing reagent kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, aminated compounds, NAD (P) H oxidase (EC 1.6.3.1); Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, aminated compounds; Reagent 3 is made up of damping fluid, stabilizing agent, NAD (P) H oxidase.Coenzyme, aminated compounds, the position of NAD (P) H oxidase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described monoamine oxidase diagnosing reagent kit of claim 2, 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 (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762309A (en) * | 2015-04-07 | 2015-07-08 | 安徽省农业科学院水稻研究所 | Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762309A (en) * | 2015-04-07 | 2015-07-08 | 安徽省农业科学院水稻研究所 | Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof |
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