CN101620084A - Monoamine oxidase diagnosis kit and method for determining monoamine oxidase activity concentration - Google Patents
Monoamine oxidase diagnosis kit and method for determining monoamine oxidase activity concentration Download PDFInfo
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- CN101620084A CN101620084A CN200810122879A CN200810122879A CN101620084A CN 101620084 A CN101620084 A CN 101620084A CN 200810122879 A CN200810122879 A CN 200810122879A CN 200810122879 A CN200810122879 A CN 200810122879A CN 101620084 A CN101620084 A CN 101620084A
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- monoamine oxidase
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Abstract
The invention relates to a monoamine oxidase diagnosis kit using the technology of an enzyme colorimetric method and an ELISA method, a method for determining monoamine oxidase activity concentration, and composition and components of a reagent, and belongs to the technical field of medical test and determination. The kit comprises the following main components: buffer solution, reduced coenzyme, amines, sodium bicarbonate (carbon dioxide), acetyl phosphate, pyruvate oxidase, malic dehydrogenase and a stabilizer. The method comprises the following steps: mixing a sample and the reagent according to a certain volume ratio to perform a series of enzymic reaction, and then placing reactants under a UV/ visible light analyzer to test the descending speed of absorbance at the position where the dominant wave length is 340nm to determine the activity concentration of the monoamine oxidase.
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-phenylethylamine), 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 (Enzymatic ColorimetricMethod) 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 reagent 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:
Aminated compounds+water+oxygen
Monoamine oxidaseCorresponding aldehyde product+ammonia+hydrogen peroxide
Carbon dioxide+acetyl phosphate+hydrogen peroxide
Pyruvate oxidasePhosphate radical+pyruvic acid+oxygen+water
Pyruvic acid+carbon dioxide+reduced coenzyme
Malic dehydrogenase (decarboxylation)Malic acid+coenzyme
This method is used monoamine oxidase (Monoamine Oxidase; EC 1.4.3.4; EC 1.4.3.6) enzyme (idol) connection pyruvate oxidase (pyruvate oxidase; EC 1.2.3.3), malic dehydrogenase (malatedehydrogenase (decarboxylating); EC 1.1.1.38; EC 1.1.1.39; EC 1.1.1.40; EC1.1.1.83) enzymatic reaction continuous monitoring method.The reaction of monoamine oxidase enzymolysis aminated compounds produces hydrogen peroxide, the effect of uniting pyruvate oxidase, malic dehydrogenase again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, 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 of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Pyruvate oxidase 10000U/L
Malic dehydrogenase 10000U/L
Aminated compounds 10mmol/L
Sodium bicarbonate (carbon dioxide) 16mmol/L
Acetyl phosphate 8mmol/L
Monoamine oxidase diagnosing reagent kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate.
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, reduced coenzyme, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate.
Reagent 2
Damping fluid, stabilizing agent, pyruvate oxidase, malic dehydrogenase.
Reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), the position of acetyl phosphate 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.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate.
Reagent 2
Damping fluid, stabilizing agent, malic dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, pyruvate oxidase.
Reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), the position of acetyl phosphate 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 reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.
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 500mmol/L
Reduced coenzyme 0.25mmol/L
Pyruvate oxidase 10000U/L
Malic dehydrogenase 10000U/L
Aminated compounds 10mmol/L
Sodium bicarbonate (carbon dioxide) 16mmol/L
Acetyl phosphate 8mmol/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 1.8 ± 0.7, 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 negative reaction (reaction descends), 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 descends, 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
Reduced coenzyme 0.25mmol/L
Aminated compounds 10mmol/L
Sodium bicarbonate (carbon dioxide) 16mmol/L
Acetyl phosphate 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Pyruvate oxidase 10000U/L
Malic dehydrogenase 10000U/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 1.8 ± 0.7, 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 negative reaction (reaction descends), 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 descends, 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
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Aminated compounds 10mmol/L
Sodium bicarbonate (carbon dioxide) 16mmol/L
Acetyl phosphate 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Malic dehydrogenase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Pyruvate oxidase 10000U/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 1.8 ± 0.7, 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 negative reaction (reaction descends), 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 descends, thereby calculates the active concentration size of monoamine oxidase.
The applicant adopts other assay methods of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.0004; Absorbance time response curve should linearly descend; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 500U/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 3%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, the linear range broadness, and stationary phase is long, is enough to easy to utilize.
Claims (6)
1. method for determining monoamine oxidase activity concentration that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Aminated compounds+water+oxygen
Monoamine oxidaseCorresponding aldehyde product+ammonia+hydrogen peroxide
Carbon dioxide+acetyl phosphate+hydrogen peroxide
Pyruvate oxidasePhosphate radical+pyruvic acid
+ oxygen+water
Pyruvic acid+carbon dioxide+reduced coenzyme
Malic dehydrogenase (decarboxylation)Malic acid+
Coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance descends, calculate the active concentration size result of monoamine oxidase.
2. monoamine oxidase diagnosing reagent kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Pyruvate oxidase 1000---80000U/L
Malic dehydrogenase 1000---80000U/L
Aminated compounds 1---50mmol/L
Sodium bicarbonate (carbon dioxide) 1---50mmol/L
Acetyl phosphate 1---50mmol/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.
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, reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate.
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, reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate oxidase, malic dehydrogenase.Reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), the position of acetyl phosphate 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, reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, sodium bicarbonate (carbon dioxide), acetyl phosphate; Reagent 2 is made up of damping fluid, stabilizing agent, malic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, pyruvate oxidase.Reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate (carbon dioxide), the position of acetyl phosphate 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 (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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| CN200810122879A CN101620084A (en) | 2008-06-30 | 2008-06-30 | Monoamine oxidase diagnosis kit and method for determining monoamine oxidase activity concentration |
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Open date: 20100106 |