CN101464290A - Monoamine oxidase diagnostic reagent kit and method for measuring active concentration of monoamine oxidase - Google Patents

Abstract

The invention relates to a kit for diagnosing monoamine oxidase by utilizing the technologies of the enzymic colorimetry and the enzyme linked immunosorbent assay. The invention further relates to a method, a principle and the composition and the components of a reagent for measuring the active concentration of the monoamine oxidase, and belongs to the technical field of medical inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, an amine compound, sodium bicarbonate, acetylcoenzyme A, pyruvate oxidase, malic dehydrogenase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, a series of enzymatic reactions occur, then the reactant is placed under an ultraviolet/visible light analyzer, and the velocity of the decrease in absorbance at 340 nm of the dominant wavelength is detected, thereby measuring the active concentration of the monoamine oxidase.

Description

Monoamine oxidase diagnosing reagent kit and method for determining monoamine oxidase activity concentration

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

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 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 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+ammonium ion+

Hydrogen peroxide

Hydrogen peroxide+carbon dioxide+acetyl coenzyme A Pyruvate oxidasePyruvic acid+

Coacetylase+oxygen

Pyruvic acid+carbon dioxide+reduced coenzyme Malic dehydrogenase (decarboxylation)

L 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 1.2.3.6), malic dehydrogenase (malatedehydrogenase; EC 1.1.1.38; EC 1.1.1.39; EC 1.1.1.40; EC 1.1.1.83) 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.

Wherein, carbon dioxide can replace with sodium bicarbonate or other supercarbonates.

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 12000U/L

Aminated compounds 9mmol/L

Sodium bicarbonate 12mmol/L

Acetyl coenzyme A 2mmol/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, acetyl coenzyme A.

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, acetyl coenzyme A.

Reagent 2

Damping fluid, stabilizing agent, pyruvate oxidase, malic dehydrogenase.

Reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate, the position of acetyl coenzyme A 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, acetyl coenzyme A.

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, the position of acetyl coenzyme A 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 6000U/L

Malic dehydrogenase 10000U/L

Aminated compounds 9mmol/L

Sodium bicarbonate 12mmol/L

Acetyl coenzyme A 2mmol/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 9mmol/L

Sodium bicarbonate 12mmol/L

Acetyl coenzyme A 2mmol/L

Reagent 2

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

Stabilizing agent 500mmol/L

Pyruvate oxidase 6000U/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 9mmol/L

Sodium bicarbonate 12mmol/L

Acetyl coenzyme A 2mmol/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 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 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)≤0.0013; Absorbance time response curve should linearly descend; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 250U/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach that 0.0003 ± 0.00015 Δ A/min/U/L---the present invention is highly sensitive, degree of accuracy good, and the linear range broadness 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+ammonium ion+

Hydrogen peroxide

Hydrogen peroxide+carbon dioxide+acetyl coenzyme A Pyruvate oxidasePyruvic acid+

Coacetylase+oxygen

Pyruvic acid+carbon dioxide+reduced coenzyme Malic dehydrogenase (decarboxylation)

L 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.

Wherein, carbon dioxide can replace with sodium bicarbonate or other supercarbonates.

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 1---50mmol/L

Acetyl coenzyme A 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: 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, acetyl coenzyme A.

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, acetyl coenzyme A; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, sodium bicarbonate, acetyl coenzyme A; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate oxidase, malic dehydrogenase.Reduced coenzyme, pyruvate oxidase, malic dehydrogenase, aminated compounds, sodium bicarbonate, the position of acetyl coenzyme A 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, acetyl coenzyme A; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, aminated compounds, sodium bicarbonate, acetyl coenzyme A; 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, the position of acetyl coenzyme A 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 (Ethyleneglycol) and at least one of the preservatives.