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Exosome purity analysis method

A technology of purity analysis and exosomes, applied in the field of analysis, can solve problems such as inaccurate results, difficulty in exosome separation, complex content components, etc., and achieve the effect of improving accuracy

Pending Publication Date: 2022-03-01
九天览月生物科技(天津)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The separation of exosomes is a new industry in recent years. The commonly used method for analyzing the purity of exosomes is the molecular exclusion method. Because the exosomes are similar in shape but different in size, and the content components are complex, in addition to exosomes, Can also contain other impurities, such as lipids and protein polymers, making exosome isolation extremely difficult
There are often some impurities in exosome samples, which may be liposomes or some impurities produced during the preparation process. If the purity of exosomes is judged only by molecular exclusion method, it is obviously possible to produce inaccurate results. result

Method used

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Experimental program
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Embodiment 1

[0032] 1. HPLC detection

[0033] Exosome sample 1 was taken and passed through a high-performance liquid chromatograph using a SEC column (Acclaim SEC-1000, 7 µm, 1000 Å, 7.8 x 150 mm) at a flow rate of 0.3ml / min, containing 150mM NaCl in 20mM phosphate ( PH=7.2) The aqueous solution is used as the mobile phase for isocratic elution. The loading volume of exosome sample 1 is 25ul, and the detection is performed at the ultraviolet absorbance value of 280nm.

[0034] According to the molecular weight of the protein marker, it is known that the peak position of exosomes is 9min and the map is processed to preliminarily determine the purity of exosomes, such as figure 1 As shown, the purity is 100%.

[0035] 2. Fluorescein sodium staining particle count detection

[0036] Take exosome sample 1 (the same sample as detected by HPLC), take 50ul into a 2ml centrifuge tube; add 200ul of saponin solution with a concentration of 10mg / ml and mix it well, and break the membrane of exoso...

Embodiment 2

[0042] 1. HPLC detection

[0043] Exosome sample 2 was taken and passed through a high-performance liquid chromatograph using an SEC column (Acclaim SEC-1000, 7 µm, 1000 Å, 7.8 x 150 mm) at a flow rate of 0.3ml / min, containing 150mM NaCl in 20mM phosphate ( PH=7.2) aqueous solution is used as the mobile phase for isocratic elution, the loading volume of exosome sample 2 is 25ul, and the detection is carried out at the ultraviolet absorbance value of 280nm. According to the molecular weight of the protein marker, it is known that the peak position of exosomes is 9min and the map is processed to preliminarily determine the purity of exosomes, such as image 3 As shown, the purity is 54.0799%.

[0044] 2. Fluorescein sodium staining particle count detection

[0045] Take exosome sample 2 (the same sample as detected by HPLC), take 50ul to a 2ml centrifuge tube; add 200ul saponin solution with a concentration of 10mg / ml and mix it well, and break the membrane of exosomes so that...

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Abstract

The invention discloses an exosome purity analysis method, a molecular exclusion method is combined with a particle counting method, in the analysis method, an SEC column is adopted to carry out molecular size purity analysis on an exosome sample, but the SEC cannot specifically separate lipidosome or protein polymer similar to the exosome in size. Therefore, particles of the dyed sample are counted in combination with a nanoflow detector, the exosomes and impurities are counted respectively according to the principle that the nanoflow detector can dye the exosomes and cannot dye lipidosome and protein, and the purity accuracy of the exosomes is improved.

Description

technical field [0001] The invention belongs to the technical field of analysis, and in particular relates to a method for analyzing the purity of exosomes. Background technique [0002] The separation of exosomes is a new industry in recent years. The commonly used method for analyzing the purity of exosomes is the molecular exclusion method. Because the exosomes are similar in shape but different in size, and the content components are complex, in addition to exosomes, It can also contain other impurities, such as lipids and protein polymers, which make the isolation of exosomes extremely difficult. There are often some impurities in exosome samples, which may be liposomes or some impurities produced during the preparation process. If the purity of exosomes is judged only by the molecular exclusion method, it is obviously possible to produce inaccurate results. result. Contents of the invention [0003] In order to solve the above technical problems, the present invent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/10G01N30/02
CPCG01N15/10G01N30/02
Inventor 葛啸虎崔明旭王娜陆路韩春乐赵刚刚
Owner 九天览月生物科技(天津)有限公司