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Monoamine oxidase activity determination method and monoamine oxidase diagnostic kit

A technology of monoamine oxidase and diagnostic kit, which is applied in the field of medical inspection and measurement, can solve the problems of difficult measurement, inability to apply automatic biochemical instrument analysis, and limited practicability, and achieve credible detection results, low test cost, and high accuracy. Effect

Inactive Publication Date: 2006-06-21
SUZHOU ANJ BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The principle of the benzylamine method is that benzylamine generates benzylaldehyde under the action of monoamine oxidase, and then reacts with dinitrophenylhydrazine under the condition of strong alkaline sodium hydroxide to generate aldehyde phenylhydrazone, which is difficult to measure on a biochemical instrument; The p-benzylamine-β-azonaphthol method needs to be extracted with cyclohexane, which limits the practicability of the method and cannot be analyzed by a fully automatic biochemical analyzer

Method used

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  • Monoamine oxidase activity determination method and monoamine oxidase diagnostic kit
  • Monoamine oxidase activity determination method and monoamine oxidase diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment one (single dose)

[0048] Prepare the monoamine oxidase activity diagnostic kit according to the following ingredients and dosage:

[0049] Glycine buffer 80mmol / l,

[0050] Butylamine 1mmol / l,

[0051] 2-ketoglutarate 3mmol / l,

[0052] thio-NADH 0.2mmol / l,

[0053] Glutamate dehydrogenase 50000U / l,

[0054] Ethylene glycol 50% (accounting for the total volume of the reagent).

[0055] Set on the automatic biochemical analyzer (Hitachi-7080): temperature 37°C, reaction time 10 minutes, test main wavelength 340nm, test secondary wavelength 405nm or more, the volume ratio of the tested monoamine oxidase sample to the reagent is 1:25, the reaction The direction is negative reaction (decrease in absorbance, the same below), the delay time is 1 minute, the detection time is 2 minutes, and the theoretical K value is -4180.

[0056] Add the sample and the prepared single agent, and the two are automatically mixed inside the analyzer, and the decrease of the a...

Embodiment 2

[0057] Embodiment two (two doses)

[0058] Prepare the monoamine oxidase activity diagnostic kit according to the following ingredients and dosage:

[0059] Reagent I——

[0060] Imidazole ~ hydrochloric acid buffer 100mmol / l,

[0061] 2-ketoglutarate 12mmol / l,

[0062] NADPH 0.25mmol / l,

[0063] Glutamate dehydrogenase 120000U / l,

[0064] Glycerol 50% (accounting for the total volume of reagent I);

[0065] Reagent II——

[0066] Imidazole ~ hydrochloric acid buffer 100mmol / l,

[0067] β-Phenylethylamine 6mmol / l,

[0068] Ethylene glycol 20mmol / l.

[0069] Set on the automatic biochemical analyzer (Hitachi-7080): temperature 30°C, reaction time 15 minutes, test main wavelength 340nm, test secondary wavelength 405nm or more, the ratio of the total volume of the tested monoamine oxidase sample to reagent I and reagent II 1:25, the ratio of reagent I to reagent II is 4:1, the reaction direction is negative reaction, the delay time is 1 minute, the detection time is 2 minu...

Embodiment 3

[0071] Embodiment three (three doses)

[0072] Prepare the monoamine oxidase activity diagnostic kit according to the following ingredients and dosage:

[0073] Reagent I

[0074] Triethanolamine buffer 120mmol / l,

[0075] 2-ketoglutarate 20mmol / l,

[0076] thio-NADPH 0.3mmol / l,

[0077] Propylene glycol 20mmol / l;

[0078] Reagent II——

[0079] Triethanolamine buffer 120mmol / l,

[0080] Glutamate dehydrogenase 200000U / l,

[0081] Propylene glycol 50% (accounting for the total volume of reagent II);

[0082] Reagent III——

[0083] Triethanolamine buffer 120mmol / l,

[0084] Tyramine 10mmol / l,

[0085] Ethylene glycol 20mmol / l.

[0086] Set on the automatic biochemical analyzer (Hitachi-7080): temperature 25°C, reaction time 20 minutes, test main wavelength 340nm, test secondary wavelength 405nm or more, the total volume of the tested sample and reagent I, reagent II and reagent III The ratio is 1:25, the amount of reagent I, reagent II and reagent III is 8:1:1, the r...

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Abstract

The invention relates measuring method of monoamine oxidase activity and its diagnostic kit. The invention comprises the following steps: using the monoamine oxidase to hydrolyze benzylamine, p-toluidine red-ª‰-azo naphthol, butyl amine, amyl amine, ª‰-phenylethylamine, tyramine and other aminated compounds to produce the ammonia, then the ammonia carried out coupling reaction with glutamate dehydrogenase to turn the oxidation type coenzyme to reduction type coenzyme, detecting the reducing velocity of absorbance of main wavelength 340nm to reflect the activity of monoamine oxidase in sample by definite quantity. The method has high specificity and good accuracy. The diagnostic kit is made to double agents or tri-agent to reduce the across impact and keep the agent stability and be good for long term storage. The method can be rapidly detected by the ultra-violet / visible light analysis meter or semi-automatic / full-automatic biochemical analysis meter, so the method is easy to spread and use.

Description

technical field [0001] The invention relates to a method for measuring monoamine oxidase activity by an enzymatic method and a monoamine oxidase diagnostic kit prepared by applying the method, which belongs to the technical field of medical inspection and measurement. Background technique [0002] In the prior art, the methods for measuring the activity of monoamine oxidase (MAO) mainly include: fluorescence method, immunosuppression method and chemical spectrophotometry. The fluorescence method uses β-phenylethylamine as a substrate to detect monoamine oxidase, and the basis is that the reaction rate of β-phenylethylamine is the highest at 10-100 mg, which is higher than that of benzylamine and 5-hydroxytryptamine. The immunological method uses monoamine oxidase antibody to react with monoamine oxidase to measure the monoamine oxidase isoenzyme formed after the reaction. Currently, the most widely used monoclonal antibodies are MAO-A3C9, MAO-A4F10, MAO-A7B10, MAO-A7E10 and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/32
Inventor 王尔中
Owner SUZHOU ANJ BIOTECHNOLOGY CO LTD
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