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Method for measuring enzyme substrate amount by combining enzyme reaction process method and terminal balance method

A technology of reaction end point and end point balance, which is applied in the preparation of test samples, color/spectral characteristic measurement, etc.

Active Publication Date: 2012-06-20
上海睿康生物科技有限公司
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Problems solved by technology

[0005] This enzymatic reaction kinetic process analysis method to determine the amount of substrate requires the reaction start signal and the reaction end signal to obtain the net change of the detection signal of the reaction system under the action of the enzyme. It predicts the end-point signal of the reaction by analyzing the data before the enzyme reaction is completed instead of directly measuring the end-point signal of the reaction

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  • Method for measuring enzyme substrate amount by combining enzyme reaction process method and terminal balance method
  • Method for measuring enzyme substrate amount by combining enzyme reaction process method and terminal balance method
  • Method for measuring enzyme substrate amount by combining enzyme reaction process method and terminal balance method

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Embodiment 1

[0034] Embodiment 1: Determination of phenol acetate by joint end-point equilibrium method and enzyme reaction kinetics process analysis method

[0035] Human serum was precipitated by 85% ammonium sulfate, the aromatic esterase in the precipitate was dissolved in 50mmol / L Tris-HCl buffer, and DEAE-cellulose ion exchange chromatography was equilibrated with the same buffer, 0.10mol / L to 1.0mol / L NaCl Concentration gradient elution and purification; phenol acetate passed through a 60-mesh silica gel column to remove impurities that inhibit human serum aromatase; after purification of human serum aromatase, phenol acetate was determined at 20U / L; its enzymatic analysis Determination of phenol acetate operating steps are as follows:

[0036] (1), add 2.0mmol / L CaCl in the test tube 2 A total of 0.90ml of 50mmol / L Tris-HCl buffer solution (pH 7.4) and 100μl of phenol acetate aqueous solution samples of different concentrations were kept in a water bath at 25°C for 5 minutes and t...

Embodiment 2

[0045] Embodiment 2: Determination of ethanol by joint end-point equilibrium method and enzyme reaction kinetics process analysis method

[0046] The alcohol dehydrogenase reaction buffer used is 0.20mol / L sodium pyrophosphate (containing 75.0mmol / L semicarbazide, pH 9.2, NAD + The final concentration is 3.0mmol / L; the detailed setting is the same as the literature Anal Sci, 2007, 23 (4): 439-444); the yeast alcohol dehydrogenase (A7011) final concentration of Sigma Company used is 50U / L; The data processing method when the reaction process analysis method predicts the absorption of the end point of the reaction is completely the same as the literature (Anal Sci, 2007, 23 (4): 439-444); the representative operation steps in its application are as follows:

[0047] (1), add the above-mentioned NAD-containing protein in the test tube + Sodium pyrophosphate buffer 1.380ml and sample 100μl, after 5 minutes at 25°C water bath, transfer the above solution as completely as possible ...

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Abstract

The invention relates to a method for measuring enzyme substrate amount by combining a terminal balance method and an enzyme reaction dynamic process to process enzyme reaction process data. The method comprises the steps: the data, which has the characteristic that the signal change between adjacent data exceeds two times of the noise of an instrument, in a continuously recorded enzyme reaction curve is valid data; detection signals are measured before little enzyme liquid is added, and an enzyme liquid dilution effect is corrected to obtain a corrected starting signal; when the number of the valid data in the enzyme reaction curve is not more than 7 or the absolute value of the difference between the corrected starting signal and the final recorded data is smaller than 50 times of the noise of the instrument, a reaction terminal signal is determined by the terminal balance method, and when the number of the valid data is more than 7 or the absolute value of the difference between the corrected starting signal and the final recorded data is larger than 50 times of the noise of the instrument and the consumption ratio of the substrate achieves requirement, the reaction terminal signal is predicted by an enzyme reaction process analysis method; and the concentration of the substrate to be measured is determined according to a standard response curve with the absolute value of the difference between the corrected starting signal and the reaction terminal signal as a measuring index. The method is applicable to irreversible enzyme reaction systems which can be continuously monitored.

Description

technical field [0001] The invention relates to a data processing method for enzymatic analysis and determination of the amount of enzyme substrates in the fields of clinical inspection, sanitary inspection, etc.; it is characterized in that the detection signal before adding the enzyme to start the reaction is an uncorrected start signal, and the tool enzyme added later is corrected. The solution dilution effect is corrected to obtain the starting point signal; according to the continuous monitoring of the effective data volume in the enzyme reaction curve, the substrate consumption ratio and the absolute value of the difference between the corrected starting point signal and the recorded final signal, choose whether to use the end point balance method or use the enzyme reaction The process analysis method analyzes the data to determine the reaction end signal, and takes the difference between the corrected start signal and the reaction end signal as the net change of the dete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N1/38
Inventor 廖飞杨晓兰龙高波刘红博廖娟
Owner 上海睿康生物科技有限公司
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