Method for detecting activity of residual trypsin in cell product

A trypsin and detection method technology, applied in the field of protein detection, can solve the problems of unsuitable detection of trypsin activity, etc., and achieve the effects of simple method, improved sensitivity, and increased reaction temperature

Inactive Publication Date: 2017-01-04
TIANJIN AMCELLGENE ENG
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Problems solved by technology

At present, the commonly used methods are all for trypsin preparations with relatively hi

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  • Method for detecting activity of residual trypsin in cell product
  • Method for detecting activity of residual trypsin in cell product
  • Method for detecting activity of residual trypsin in cell product

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[0023] A method for detecting residual trypsin activity in cell products, comprising the following steps:

[0024] (1) Blank experiment: Take 2.5-3.5ml of the substrate solution, add 150-250μl of 0.001mol / L hydrochloric acid solution, mix well, and use it as a blank experiment.

[0025] (2) Determination of residual trypsin activity of cell products: take 100 μl of cell product solution, add 2.5-3.5 ml of substrate solution and 80-120 μl of phosphate buffer solution, immediately time and mix evenly, and measure the absorbance OD value at 253nm A2, while keeping the reaction system at 36-38°C; then measure the absorbance OD value A1 at 253nm at the point of reaction for 25 minutes; A1-A2 represent the residual trypsin activity in the product.

[0026] In order to further optimize the above technical scheme, take 3.0ml of the substrate solution, add 200μl of 0.001mol / L hydrochloric acid solution, mix well, and use it as a blank experiment; the blank experiment is used to offset ...

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Abstract

The invention discloses a method for detecting the activity of residual trypsin in a cell product. The method is characterized in that trypsin is used to catalyze hydrolysis of N-benzoyl-L-arginine ethyl ester hydrochloride into N-benzoyl-L-arginine hydrochloride and ethanol, and detecting the absorbance of the hydrolyzed reaction system at a wavelength of 253 nm in order to determine the activity of trypsin. The new trypsin activity detection method is obtained when the optimum reaction temperature is 37 DEG C and the optimum detection time is 25 min after the reaction starts; and the method has high sensitivity, so the method can be used to detect the activity of the residual trypsin in the cell product. The method for detecting the activity of residual trypsin in the cell product is simple, sensitive and practical.

Description

technical field [0001] The invention relates to the technical field of protein detection, in particular to a method for detecting residual trypsin activity in cell products. Background technique [0002] Trypsin is an essential reagent in the cell culture process and is used for the digestion of adherent cells. However, when the cell products are used in the human body, the residual trypsin activity may cause potential harm to the user. Therefore, residual trypsin activity in cell preparations should be detected and limited. [0003] As a proteolytic enzyme, trypsin has high specificity for the bonds formed by the carboxyl groups of basic amino acids (such as arginine and lysine) and the amino groups of other amino acids; The amide bond or ester bond formed by the carboxyl group; therefore, the activity of trypsin can be determined by using the amide or ester compound containing these bonds as a substrate. Currently commonly used methods are aimed at trypsin preparations w...

Claims

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Application Information

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IPC IPC(8): C12Q1/37C12Q1/02
Inventor 韩忠朝王斌冯杰韩之波耿洁
Owner TIANJIN AMCELLGENE ENG
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