CN101793719A - Monoamine oxidase diagnostic reagent (kit) and method for determining activity concentration of monoamine oxidase - Google Patents

Abstract

The invention relates to a monoamine oxidase diagnosing/determining reagent (kit) utilizing the technologies of the indirect enzymatic recycling amplification method, 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 reduced coenzyme, an amine compound, oxidized ferredoxin, a ferredoxin nitrite reductase, a nitrite reductase 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

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 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-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 indirect enzyme cycle amplification method (IndirectEnzymatic Recycling Method) of utilizing, enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) 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 in order to the monoamine oxidase diagnostic of realizing this method/mensuration reagent (box), 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+2 water+6 oxidized form ferredoxin Ferredoxin-nitrite reductase

Nitrous acid+6 reduced form ferredoxins

Nitrous acid+3 reduced coenzymes Nitrite reductaseAmmonium hydroxide+3 coenzyme+water

This method is used monoamine oxidase (Monoamine Oxidase; EC 1.4.3.4; EC 1.4.3.6) enzyme (idol) connection ferredoxin-nitrite reductase (ferredoxin-nitrite reductase; EC 1.7.7.1), nitrite reductase (nitrite reductase; EC 1.7.1.4) enzymatic reaction continuous monitoring method.Monoamine oxidase and ferredoxin-nitrite reductase are as the effect enzyme, and function is that the aminated compounds enzymolysis is produced nitrous acid.Nitrite reductase is as cyclophorase: become nitrous acid again ammonia (ammonium hydroxide) once more, it is recycling that ammonia constantly is repeated.Nitrite reductase is as the colour developing enzyme, reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into oxidized coenzyme (not having absorption peak 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

Ferredoxin-nitrite reductase 10000U/L

Nitrite reductase 10000U/L

Aminated compounds 3mmol/L

Oxidized form ferredoxin 5mmol/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, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, oxidized form ferredoxin.

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, oxidized form ferredoxin.

Reagent 2

Damping fluid, stabilizing agent, ferredoxin-nitrite reductase, nitrite reductase.

Reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, the position of oxidized form ferredoxin 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, oxidized form ferredoxin.

Reagent 2

Damping fluid, stabilizing agent, nitrite reductase.

Reagent 3

Damping fluid, stabilizing agent, ferredoxin-nitrite reductase.

Reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, the position of oxidized form ferredoxin 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

Ferredoxin-nitrite reductase 10000U/L

Nitrite reductase 10000U/L

Aminated compounds 3mmol/L

Oxidized form ferredoxin 5mmol/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

Aminated compounds 3mmol/L

Oxidized form ferredoxin 5mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Ferredoxin-nitrite reductase 10000U/L

Nitrite reductase 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

Aminated compounds 3mmol/L

Oxidized form ferredoxin 5mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Nitrite reductase 10000U/L

Reagent 3

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

Stabilizing agent 500mmol/L

Ferredoxin-nitrite reductase 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)≤0.0001; Absorbance time response curve should linearly descend; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 400U/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 0.0010 ± 0.0005 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 indirect enzyme cycle amplification 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+2 water+6 oxidized form ferredoxin Ferredoxin-nitrite reductase

Nitrous acid+6 reduced form ferredoxins

Nitrous acid+3 reduced coenzymes Nitrite reductaseAmmonium hydroxide+3 coenzyme+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. monoamine oxidase diagnostic/mensuration reagent (box), principal ingredient comprises:

Damping fluid 20---500mmol/L

Stabilizing agent 1---4000mmol/L

Reduced coenzyme 0.1---0.35mmol/L

Ferredoxin-nitrite reductase 1000---80000U/L

Nitrite reductase 1000---80000U/L

Aminated compounds 1---50mmol/L

Oxidized form ferredoxin 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, reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, oxidized form ferredoxin.

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, reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, oxidized form ferredoxin; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, oxidized form ferredoxin; Reagent 2 is made up of damping fluid, stabilizing agent, ferredoxin-nitrite reductase, nitrite reductase.Reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, the position of oxidized form ferredoxin 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, reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, oxidized form ferredoxin; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, oxidized form ferredoxin; Reagent 2 is made up of damping fluid, stabilizing agent, nitrite reductase; Reagent 3 is made up of damping fluid, stabilizing agent, ferredoxin-nitrite reductase.Reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, aminated compounds, the position of oxidized form ferredoxin 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: glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.