CN101762480A - Monoamine oxidase diagnostic reagent (kit) and activity concentration determining method of monoamine oxidase - Google Patents

Abstract

The invention relates to a monoamine oxidase diagnostic reagent (kit) utilizing the techniques of an enzyme multiplying method, an enzyme colorimetric method and an enzyme coupling method, and a method for determining the activity concentration of monoamine oxidase as well as composition and components of the reagent, belonging to the technical field of the detection and determination of medicine. The reagent (kit) mainly comprises the components of a buffer solution, reduced coenzyme, amine compounds, pyruvic acid, pyruvic acid dehydrogenase, NADH peroxidase and a stabilizer. The method comprises the following steps of: mixing a sample and the reagent according to a certain volume ratio to perform a series of enzymic reactions, then placing a reactant under an ultraviolet/visible light analyzer, detecting the declining speed of absorbance in the position of 340nm of main wave length, and determining the activity concentration of the monoamine oxidase.

Description

Monoamine oxidase diagnosing reagent (box) and method for determining monoamine oxidase activity concentration

Technical field

The present invention relates to a kind of monoamine oxidase diagnosing 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-A7E10 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 enzyme multiplication method (Enzymatic DoublingMethod) that utilizes, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (CoupleReaction) technology, 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 the monoamine oxidase diagnosing reagent (box) in order to realize 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 of the present invention is as follows:

Aminated compounds+water+oxygen Monoamine oxidaseCorresponding aldehyde product+ammonia+hydrogen peroxide

Ammonia+pyruvic acid+reduced coenzyme Alanine dehydrogenaseAlanine+water+coenzyme

Hydrogen peroxide+reduced coenzyme The NADH peroxidaseCoenzyme+2 water

This method is used monoamine oxidase (Monoamine Oxidase; EC 1.4.3.4; EC 1.4.3.6) enzyme (idol) connection alanine dehydrogenase (alanine dehydrogenase; EC 1.4.1.1), NADH peroxidase (NADH peroxidase; EC 1.11.1.1; EC 1.11.1.2) enzymatic reaction continuous monitoring method.The reaction of monoamine oxidase enzymolysis aminated compounds produces ammonia and hydrogen peroxide, the effect of uniting alanine dehydrogenase, NADH peroxidase more respectively by (idol), final secondary is oxidized into coenzyme (not having absorption peak at the 340nm place) with reduced coenzyme (absorption peak being arranged at the 340nm place), 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 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

Reduced coenzyme 0.25mmol/L

Alanine dehydrogenase 10000U/L

NADH peroxidase 10000U/L

Aminated compounds 12mmol/L

Pyruvic acid 15mmol/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, reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, pyruvic acid.

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, reduced coenzyme, aminated compounds, pyruvic acid.

Reagent 2

Damping fluid, stabilizing agent, alanine dehydrogenase, NADH peroxidase.

Reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, the position of pyruvic acid 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, reduced coenzyme, aminated compounds, pyruvic acid.

Reagent 2

Damping fluid, stabilizing agent, NADH peroxidase.

Reagent 3

Damping fluid, stabilizing agent, alanine dehydrogenase.

Reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, the position of pyruvic acid 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 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 diagnostic of present embodiment/mensuration reagent is single reagent, comprising:

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Alanine dehydrogenase 10000U/L

NADH peroxidase 10000U/L

Benzylamine 12mmol/L

Pyruvic acid 15mmol/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 diagnostic of present embodiment/mensuration reagent is double reagent, comprising:

Reagent 1

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Benzylamine 12mmol/L

Pyruvic acid 15mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Alanine dehydrogenase 10000U/L

NADH peroxidase 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 diagnostic of present embodiment/mensuration reagent is three reagent, comprising:

Reagent 1

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Benzylamine 12mmol/L

Pyruvic acid 15mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

NADH peroxidase 10000U/L

Reagent 3

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Alanine dehydrogenase 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)≤0.001; Absorbance time response curve should linearly descend; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 600U/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); The sensitivity of reagent can reach 0.0005 ± 0.0003 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 enzyme multiplication method, enzymic colorimetric and enzyme-linked method technology, its method is as follows:

Aminated compounds+water+oxygen Monoamine oxidaseCorresponding aldehyde product+ammonia+hydrogen peroxide

Ammonia+pyruvic acid+reduced coenzyme Alanine dehydrogenaseAlanine+water+coenzyme

Hydrogen peroxide+reduced coenzyme The NADH peroxidaseCoenzyme+2 water

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. a monoamine oxidase diagnosing reagent (box), principal ingredient comprises:

Damping fluid 20---500mmol/L

Stabilizing agent 1---4000mmol/L

Reduced coenzyme 0.1---0.35mmol/L

Alanine dehydrogenase 1000---80000U/L

NADH peroxidase 1000---80000U/L

Aminated compounds 1---50mmol/L

Pyruvic acid 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 diagnosing reagent of claim 2 (box), it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, pyruvic acid.

4. according to the described monoamine oxidase diagnosing reagent of claim 2 (box), it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, pyruvic acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, alanine dehydrogenase, NADH peroxidase.Reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, the position of pyruvic acid in reagent 1 or reagent 2 can not limit.

5. according to the described monoamine oxidase diagnosing reagent of claim 2 (box), it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, pyruvic acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, NADH peroxidase; Reagent 3 is made up of damping fluid, stabilizing agent, alanine dehydrogenase.Reduced coenzyme, alanine dehydrogenase, NADH peroxidase, aminated compounds, the position of pyruvic acid in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described monoamine oxidase diagnosing 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: glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.