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Method and kit for investigating adenosine deamiase by coupling enzymatic reaction

A technology of adenosine deaminase and adenosine deamination, which is applied in biological testing, microbial determination/inspection, biochemical equipment and methods, etc. The effect of inosin coupling enzymatic reaction system

Inactive Publication Date: 2005-11-09
ZHEJIANG YAKE SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the choice of hydrogen donor is too narrow, only EHSPT is used

Method used

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  • Method and kit for investigating adenosine deamiase by coupling enzymatic reaction
  • Method and kit for investigating adenosine deamiase by coupling enzymatic reaction
  • Method and kit for investigating adenosine deamiase by coupling enzymatic reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0034] 1. Determine the Adenosine Michaelis constant Km

[0035] 1) Reagent composition

[0036] Reagent I Trizma-base buffer pH 7.6 50 mmol / L (mmol / L)

[0037] 4-AA 2mmol / L

[0038] ADA 300 units / liter [U / L (beef intestines)]

[0039] PNP 100U / L

[0040] XOD 200U / L

[0041] POD 600U / L

[0042] Reagent II Tris-HCl buffer pH 4.0 50mmol / L

[0043] Adenosine 0.001-10mmol / L

[0044] TOOS 2mmol / L

[0045] 2) Test parameters and operation steps

[0046] Temperature 37°C

[0047] Wavelength 550 nanometers (nm)

[0048] Cuvette diameter 1.0 cm (cm)

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Abstract

Heinz(1980) makes use of the adenosine ammonia-deleted enzyme to change adenosine into hypo-yellow and gains H2O2 through the reaction of the enzyme1 and enzyme2. With the effect of the catalase and aldehyde hydrogen-deleted enzyme, Heinz measures the velocity of increase of the degree of NADPH absorbing light at 334 nms to gain adenosine ammonia-deleted enzyme's activity. The method's weakness is high cost of the reagents. The invention introduces Trinder's reaction to combine aniline type hydrogen and 4- to from a coloured product with the effect of H2O2 gained by the reaction of enzyme3 in the situation of the catalase. By watching the quantity of this coloured product, people can determine the adenosine ammonia-deleted enzyme's activity. The invention simplifies the Heinz's reaction system and lowers the cost. The invention uses the aniline compound ADOS, ADPS, ALPS, TODB, TOOS and TOPS as hydrogen provider for Trinder's reaction and increases the reaction's agility. The invention also involves a reagent box used for implementing the method mentioned above.

Description

technical field [0001] The invention relates to a technical scheme for measuring the activity of adenosine deaminase in biological samples and a reagent produced by the scheme. In particular, it relates to a multi-enzyme-catalyzed reaction system coupled with adenosine deaminase to generate inosine from adenosine deamination. Background technique [0002] Heinz (1980) deaminated adenosine (Adenosine) to produce inosine (Inosine) by means of adenosine deaminase (Adenosine deaminase, ADA). Hydrogen peroxide (H 2 o 2 ). Catalase reduces hydrogen peroxide to water, and generates acetaldehyde in the presence of ethanol. Acetaldehyde depends on NADP + Oxidized by aldehyde dehydrogenase (Aldehyde dehydrogenase), while NADP + is reduced to NADPH. ADA activity was measured by measuring the rate of increase in NADPH absorbance at 334 nm. The disadvantage of this method is that the reaction system is too cumbersome and the cost of reagents is high, which hinders the clinical ap...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 蔡枫
Owner ZHEJIANG YAKE SCI & TECH