CN101793714A - Monoamine oxidase diagnostic reagent (kit) and method for determining activity concentration of monoamine oxidase - Google Patents
Monoamine oxidase diagnostic reagent (kit) and method for determining activity concentration of monoamine oxidase Download PDFInfo
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- CN101793714A CN101793714A CN200910028227A CN200910028227A CN101793714A CN 101793714 A CN101793714 A CN 101793714A CN 200910028227 A CN200910028227 A CN 200910028227A CN 200910028227 A CN200910028227 A CN 200910028227A CN 101793714 A CN101793714 A CN 101793714A
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- galactose
- monoamine oxidase
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Abstract
The invention relates to a monoamine oxidase diagnosing/determining reagent (kit) utilizing the technologies of the enzymatic-colorimetric method and the enzyme-link method and simultaneously also relates to a method, constitution and components of a reagent for determining the activity concentration of the monoamine oxidase, belonging to the technical field of medical testing and determination. The reagent (kit) comprises the following principle components: a buffer solution, a coenzyme, an amine compound, galactose hexose dialdehyde aldose, a galactose oxidase, a galactose dehydrogenase and a stabilizer; and samples and the reagent are mixed according to a certain volume ratio so as to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet/visible light analyzer so as to detect the ascending velocity of absorbency of a part with dominant wavelength being 340 nm, thereby obtaining the activity concentration of the monoamine oxidase through measurement and calculation.
Description
Technical field
The present invention relates to a kind of monoamine oxidase diagnostic/mensuration reagent (box), the invention still further relates to the method for measuring monoamine oxidase activity concentration simultaneously, belong to medical test determination techniques field.
Background technology
In the prior art, measure the active method of monoamine oxidase (MAO) and mainly contain: fluorescence method, immunodepression and chemical spectrophotometric method.Fluorescence method is to detect monoamine oxidase with β-phenyl ethylamine as substrate, and it is according to being: β-phenyl ethylamine is the reaction velocity maximum when 10~100mg, is higher than the reaction velocity of benzylamine and serotonin.Immunological method then utilizes monoamine oxidase antibody and monoamine oxidase to react, the monoamine oxidase isodynamic enzyme that forms behind the assaying reaction, using more monoclonal antibody at present has MAO-A3C9, MAO-A4F10, MAO-A7B10, MAO-A7 carbamyl phosphate synthetase 0 and MAO-B1C2 etc.
Comparatively speaking, chemical spectrophotometric method should have more general.Hydrogen peroxide (the H that can discharge with monoamine oxidase catalysis amine
2O
2) deoxidation 10-N-methylamino formoxyl-3; 7-dimethylamino-10-hydrogen-phenothiazine colour formers such as (MCDP) is measured; also can use benzylamine (Benzylamine), P-Toluidine-β-azo naphthols, butylamine (butylamine), amylamine (amylamine), β-phenethyl amine (b-phenylethylamine), tyrasamine (tyramine; claim again " 3-Uteramin ", 3-parahdroxyphenylethylamine), serotonin (5-hydroxytryptamine) etc. measures the activity of monoamine oxidase as reaction substrate.Wherein, use other substrate height of specific activity of the monoamine oxidase that benzene methanamine type substrate records, make 30% of substrate and be about the 3-Uteramin with butylamine, amylamine, activity that β-phenethyl amine records as substrate.At present, monoamine oxidase is measured benzylamine and the P-Toluidine-β-azo naphthols used as substrate more.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, this measures difficult on biochemical instruments; P-Toluidine-β-azonaphthalene phenol method then needs to extract with cyclohexane, has limited the practicality of this method, and can not use the full automatic biochemical apparatus analysis.
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 the monoamine oxidase diagnostic of realizing this method/mensuration reagent (box), adopt this reagent (box) 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 of the present invention is as follows:
Aminated compounds+water+oxygen
Monoamine oxidaseCorresponding aldehyde product+ammonia+hydrogen peroxide
Hydrogen peroxide+galactose hexose dialdehyde aldose
Galactose oxidaseGalactose+oxygen
Galactose+coenzyme
Galactose dehydrogenaseGalactonic acid-1,4-lactone+reduced coenzyme
This method is used monoamine oxidase (Monoamine Oxidase; EC 1.4.3.4; EC 1.4.3.6) enzyme (idol) connection galactose oxidase (galactose oxidase; EC 1.1.3.9), galactose dehydrogenase (galactose1-dehydrogenase; EC 1.1.1.48; EC 1.1.1.120) enzymatic reaction continuous monitoring method.The reaction of monoamine oxidase enzymolysis aminated compounds produces hydrogen peroxide, the effect of uniting galactose oxidase, galactose dehydrogenase again by (idol), 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 monoamine oxidase diagnostic of the present invention/the mensurations reagent of following composition relation is ideal comparatively:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Galactose oxidase 6000U/L
Galactose dehydrogenase 8000U/L
Aminated compounds 5mmol/L
Galactose hexose dialdehyde aldose 9mmol/L
The present invention measures in the middle of the composition of diagnostic kit of activity of monoamine oxidase, one of described " aminated compounds " preferred following material: the derivant of benzylamine, P-Toluidine-β-azo naphthols, butylamine, amylamine, β-phenethyl amine, tyrasamine, serotonin or these seven kinds of materials, but range of choice be not subjected to these enumerate limit.
Monoamine oxidase diagnostic of the present invention/mensuration reagent (box) can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, galactose hexose dialdehyde aldose.
Reagent (box) can be 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, galactose hexose dialdehyde aldose.
Reagent 2
Damping fluid, stabilizing agent, galactose oxidase, galactose dehydrogenase.
Coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, the position of galactose hexose dialdehyde aldose in reagent 1 or reagent 2 can not limit.Reagent (box) can be 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, coenzyme, aminated compounds, galactose hexose dialdehyde aldose.
Reagent 2
Damping fluid, stabilizing agent, galactose dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, galactose oxidase.
Coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, the position of galactose hexose dialdehyde aldose in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be 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 diagnostic of present embodiment/mensuration reagent is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Galactose oxidase 6000U/L
Galactose dehydrogenase 8000U/L
Benzylamine 5mmol/L
Galactose hexose dialdehyde aldose 9mmol/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 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 diagnostic of present embodiment/mensuration reagent is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Benzylamine 5mmol/L
Galactose hexose dialdehyde aldose 9mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Galactose oxidase 6000U/L
Galactose dehydrogenase 8000U/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 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 diagnostic of present embodiment/mensuration reagent is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Benzylamine 5mmol/L
Galactose hexose dialdehyde aldose 9mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Galactose dehydrogenase 8000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Galactose 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≤0.1, 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.
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)≤0.001; Absorbance time response curve should linearly rise; 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 5%; The coefficient of variation (CV)≤2.5% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.0003 ± 0.00015 Δ A/min/U/L; 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 is as follows:
Aminated compounds+water+oxygen
Monoamine oxidaseCorresponding aldehyde product+ammonia+hydrogen peroxide
Hydrogen peroxide+galactose hexose dialdehyde aldose
Galactose oxidaseGalactose+oxygen
Galactose+coenzyme
Galactose dehydrogenaseGalactonic acid-1,4-lactone+reduced 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 rises, calculate the active concentration size measurement result of monoamine oxidase.
2. monoamine oxidase diagnostic/mensuration reagent (box), principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Galactose oxidase 1000---80000U/L
Galactose dehydrogenase 1000---80000U/L
Aminated compounds 1---50mmol/L
Galactose hexose dialdehyde aldose 1---50mmol/L
The present invention measures in the middle of the composition of diagnostic kit of activity of monoamine oxidase, one of described " aminated compounds " preferred following material: the derivant of benzylamine, P-Toluidine-β-azo naphthols, butylamine, amylamine, β-phenethyl amine, tyrasamine, serotonin or these seven kinds of materials, but range of choice be not subjected to these enumerate limit.
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: reagent (box) can be 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 diagnostic of claim 2/mensuration reagent (box), it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, galactose hexose dialdehyde aldose.
4. according to the described monoamine oxidase diagnostic of claim 2/mensuration reagent (box), it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, galactose hexose dialdehyde aldose; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, aminated compounds, galactose hexose dialdehyde aldose; Reagent 2 is made up of damping fluid, stabilizing agent, galactose oxidase, galactose dehydrogenase.Coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, the position of galactose hexose dialdehyde aldose in reagent 1 or reagent 2 can not limit.
5. according to the described monoamine oxidase diagnostic of claim 2/mensuration reagent (box), it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, galactose hexose dialdehyde aldose; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, aminated compounds, galactose hexose dialdehyde aldose; Reagent 2 is made up of damping fluid, stabilizing agent, galactose dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, galactose oxidase.Coenzyme, galactose oxidase, galactose dehydrogenase, aminated compounds, the position of galactose hexose dialdehyde aldose in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described monoamine oxidase diagnostic of claim 2/mensuration reagent (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 publication date: 20100804 |