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Method for testing dimethyl arginine dimethylamine hydrolytic enzyme and diagnostic reagent thereof

A technology of dimethylarginine dimethylamine and dimethylarginine, which is applied in the field of medical testing and determination, and can solve the problems of long time consumption, limitation, inability to automate the test and the like

Inactive Publication Date: 2014-03-26
ZHEJIANG YATAI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common features of the above methods are that special measuring instruments are required, the operation is complicated, the test takes a long time, it cannot be applied to the automatic analysis of clinical large flow, and it is expensive
[0005] Y-L Tain and C Baylis reported in Kidney Int.2007 October; 72(7):886-889 an improved method for the determination of DDAH in rat kidney tissue samples, which uses a manual method to determine the produced L-citrulline , low sensitivity, cannot be automated
[0006] Due to the very low content of DDAH in tissue samples, conventional clinical enzymology assay methods cannot achieve the required assay sensitivity, which limits the adoption of conventional methods
In terms of enzymatic method for measuring DDAH technology, although the signal increment technology of enzymatic detection has been continuously developed in recent years, there have been no methodological reports on the determination of DDAH activity by enzymatic cycling; there have also been no reports and reports on fully automatic diagnostic reagents for DDAH. application

Method used

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  • Method for testing dimethyl arginine dimethylamine hydrolytic enzyme and diagnostic reagent thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: The reduced coenzyme I produced by the reagent system is directly measured using the enzyme cycle reaction mechanism established by the present invention.

[0025] Reagent components

Embodiment 2

[0026] Example 2: The reagents in the above-mentioned Example 1 can also be formulated into double reagents. The position of each reagent component in reagent 1 or reagent 2 is not limited. It can be a dry powder reagent, which can be dissolved with water before use, or it can be a liquid Reagents, used directly or mixed with:

[0027] Reagent 1

[0028] Reagent 2

[0029] carbamoyl phosphate

Embodiment 3

[0030] Example 3: The reagents in the above-mentioned Example 1 can also be prepared into three reagents, the position of each reagent component in reagent 1, reagent 2 or reagent 3 is not limited, and each component can be a dry powder reagent, which is dissolved in water before use Use, also liquid reagents, straight or mixed with:

[0031] Reagent 1

[0032] Reagent 2

[0033] Reagent 3

[0034] The molar concentration of the stabilizing agent in the above-mentioned formulas of Examples 1 to 3 is 0.1 to 3000 mmol / L. When measuring samples, the fixed time method is adopted, and the ratio of reagents to samples is not fixed, but it should be consistent within the same measurement batch. For example, the reagent: sample ratio is 300:30, the temperature is 37°C, and the measurement wavelength is 340nm. After adding the sample or calibrator to the reagent, incubate at the measurement temperature for 120-300 seconds to remove the interference of albu...

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Abstract

The invention relates to a zymologic method for circularly testing the activity of dimethyl arginine dimethylamine hydrolytic enzyme, and a diagnostic reagent thereof. In a designed product detection method, hydrolysis of asymmetric dimethyl arginine is catalyzed by dimethyl arginine dimethylamine hydrolytic enzyme in a tissue sample to generate citrulline; in a novel design method, citrulline is made to continuously react with an enzyme circular reaction system consisting of citrulline hydrolytic enzyme (EC 3.5.1.20), ornithine carbamoyltransferase (EC 2.1.3.3) and a substrate of the ornithine carbamoyltransferase, so that the reaction sensitivity is greatly enhanced, and the yield of the citrulline is proportional to the activity of the dimethyl arginine dimethylamine hydrolytic enzyme in the sample; and the aim of testing the activity of the dimethyl arginine dimethylamine hydrolytic enzyme in the sample can be fulfilled by testing the generating rate of ammonia in the circular enzyme reaction system. A reagent can be a dry powder reagent, a liquid reagent, a single-dose reagent, a double-dose agent or a multi-dose agent.

Description

Technical field: [0001] The invention relates to an enzymatic cycle assay method for dimethylarginine dimethylamine hydrolase (Dimethylarginine dimethylaminohydrolase EC3.5.3.18, DDAH) activity and a diagnostic reagent thereof, belonging to the technical field of medical examination and assay. Background technique: [0002] Asymmetric dimethylarginine, a methylated arginine, is an endogenous nitric oxide synthase (Nitric oxide systhase, NOS) inhibitor, which can competitively inhibit nitric oxide (Nitric oxide, NO) Synthesis. It is an asymmetric dimethylarginine produced by intracellular protein methylation under the action of arginine methyltransferase and then hydrolysis. Dimethylarginine dimethylaminohydrolase affects the activity of NOS by regulating the level of asymmetric dimethylarginine in the body, and then regulates the production of NO in the body. There is a close relationship between the activity of DDAH and a variety of cardiovascular diseases, and it is cons...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/34C12Q1/48C12Q1/32
Inventor 王学忠
Owner ZHEJIANG YATAI PHARMA
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