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Detection method of biological activity of vascular endothelial growth factors and application

A biologically active, vascular endothelial technology, applied in the field of biological product quality detection and analysis, can solve problems affecting the repeatability and reproducibility of alamar blue, and achieve the effect of ensuring stability

Active Publication Date: 2017-11-07
TOT BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although alamar blue has been widely used in cell proliferation experiments, factors such as cell type, cell density, cell viability, cell proliferation rate, cell culture medium (such as serum concentration) often affect the repeatability and reproducibility of alamar blue detection
At present, there is no literature report on the application of alamar blue to the detection of the biological activity of VEGF, but how to use alamar blue to truly and accurately reflect the cell proliferation-promoting activity of VEGF is a technical difficulty that needs to be solved urgently in the present invention

Method used

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  • Detection method of biological activity of vascular endothelial growth factors and application
  • Detection method of biological activity of vascular endothelial growth factors and application
  • Detection method of biological activity of vascular endothelial growth factors and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Quality control is carried out before using different batches of vascular endothelial growth factor purchased, which specifically includes the following steps:

[0079] 1) subpackaging and storing the vascular endothelial growth factor freeze-dried powder;

[0080] The freeze-dried powder of vascular endothelial growth factor was transferred from -20°C to 4°C and equilibrated overnight, and then transferred from 4°C to room temperature for 2 hours, and added to the freeze-dried powder of vascular endothelial growth factor without Bacterial water was dissolved, 25 ° C water bath for 15 min, during which time every 5 min was taken out and inverted to mix, and the dissolved vascular endothelial growth factor was divided into packages, and the tube endothelial growth factor solution was placed at room temperature for 5 min in stages, and then transferred to 4 ° C to balance overnight. Then transfer to -20°C for storage;

[0081] 2) Activity detection of vascular endothelia...

Embodiment 2

[0090] The quality control of vascular endothelial growth factor before use includes the following steps:

[0091] 1) subpackaging and storing the vascular endothelial growth factor freeze-dried powder;

[0092] The freeze-dried powder of vascular endothelial growth factor was transferred from -20°C to 4°C and equilibrated overnight, and then transferred from 4°C to room temperature for 3 hours, and added to the freeze-dried powder of vascular endothelial growth factor without Bacterial water was dissolved, 23 ° C water bath for 16 min, during which every 6 min was taken out and mixed upside down, the dissolved vascular endothelial growth factor was divided into packages, and the tube endothelial growth factor solution was placed at room temperature for 6 min in stages, and then transferred to 4 ° C to balance overnight. Then transfer to -20°C for storage;

[0093] 2) Activity detection of vascular endothelial factor:

[0094] (1) Human umbilical vein endothelial cells were ...

Embodiment 3

[0102] The quality control of vascular endothelial growth factor before use includes the following steps:

[0103] 1) subpackaging and storing the vascular endothelial growth factor freeze-dried powder;

[0104] The freeze-dried powder of vascular endothelial growth factor was transferred from -20°C to 4°C to equilibrate overnight, and then transferred from 4°C to room temperature for 4 hours, and added to the freeze-dried powder of vascular endothelial growth factor Bacterial water was dissolved, 28 ° C water bath for 12 min, during which every 4 min was taken out and mixed upside down, the dissolved vascular endothelial growth factor was divided into packages, and the tube endothelial growth factor solution was placed at room temperature for 6 min in stages, and then transferred to 4 ° C to balance overnight. Then transfer to -20°C for storage;

[0105] 2) Activity detection of vascular endothelial factor:

[0106] (1) Human umbilical vein endothelial cells were cultured t...

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Abstract

The invention discloses a detection method of the biological activity of vascular endothelial growth factors. The method comprises the following steps of (1) adding trypan blue into collected human umbilical vein endothelial cells; performing counting after the uniform mixing; regulating the cell concentration to (2 to 10)*10<4>mL according to the counting result; (2) culturing the regulated cells for 3 to 10h in a CO2 culture box; (3) performing concentration gradient dilution on the endothelial growth factors; adding all gradient dilution endothelial growth factor solutions into cells cultured in the step (2); after the uniform mixing, performing culture for 80 to 100h in the CO2 culture box; (4) adding Alma blue color developing liquid into a mixed solution of the vascular endothelial growth factors and the cells cultured in the step (3); after uniform mixing, performing culturing for 1 to 10h in the CO2 culture box; (5) performing fluorescence intensity detection on the sample cultured in the step (4). Through the activity detection on the vascular endothelial growth factors, the use quantity of the vascular endothelial growth factors in different batches is regulated, so that the activity detection result is stable and more accurate.

Description

technical field [0001] The invention belongs to the field of biological product quality detection and analysis, and relates to a detection method and application of the biological activity of vascular endothelial growth factor. Background technique [0002] Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor specific to vascular endothelial cells, which can induce angiogenesis in vivo and promote angiogenesis. It is widely used in the evaluation and detection of tumor drugs, and its quality is directly related to Accuracy of drug activity test results. [0003] At present, the quality of VEGF products on the market is uneven, and there are large differences between batches, mainly because improper storage conditions and usage methods have seriously affected the biological activity of VEGF products. Therefore, in order to ensure that different VEGF products have similar biological activities, it is necessary to establish an activity control and detec...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/573G01N33/532G01N21/64
CPCG01N21/6486G01N33/532G01N33/573G01N33/6863G01N2333/4706
Inventor 张妍孙菁相凤阳程欣张贵亚
Owner TOT BIOPHARM CO LTD
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