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Rapid and high-purity separation method of human plasma exosome

A separation method, human plasma technology, applied in the direction of material separation, analysis materials, measuring devices, etc., can solve the problems of low purity, low yield, and reduce the purity of exosomes

Inactive Publication Date: 2021-08-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these technologies can obtain exosomes from plasma, the shortcomings of low purity, long separation time, and low yield limit their further development.
In particular, there is a large amount of lipoproteins in plasma, which seriously reduces the purity of exosomes obtained by most current isolation methods

Method used

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  • Rapid and high-purity separation method of human plasma exosome
  • Rapid and high-purity separation method of human plasma exosome
  • Rapid and high-purity separation method of human plasma exosome

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Experimental program
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Effect test

Embodiment 1

[0022] The agarose gel column series high performance liquid phase exclusion chromatography of the present invention is used for rapid and high-purity separation of human plasma exosomes, and the specific steps are as follows:

[0023] (1) Plasma acquisition: centrifuge the human blood collected with EDTA collection tube at 500g for 8min, and take the upper layer liquid, which is the human plasma sample, the volume is 2mL;

[0024] (2) Plasma pretreatment: centrifuge the human plasma collected in (1) at 12000g for 15min, discard the precipitate, take the upper liquid, and then filter it with a 0.22um filter membrane;

[0025] (3) Filling of agarose gel column: absorb 12mL of purchased 4B or CL-4B agarose ethanol solution, replace it with PBS solution 3 times, then pour it into a gravity flow empty tube (total volume of 12mL) , after the agarose settles freely, open the lower end of the flow tube to let the solution flow out, then push the upper sieve plate into the flow tube w...

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Abstract

The invention belongs to the technical field of liquid chromatographic separation, and particularly relates to a rapid and high-purity separation method of human plasma exosome. The method comprises the steps of firstly, removing apoptotic bodies and microvesicles of human plasma through high-speed centrifugation, then loading a sample to an agarose gel small column for pre-separation, and removing most of abundance proteins with relatively small sizes, such as human serum albumin and immune globulin; sequentially carrying out merging and ultrafiltration concentration on the collected fractions, and finally carrying out high performance liquid volume exclusion chromatography separation to realize separation of exosome and interference lipoprotein, thereby finally obtaining the high-purity human plasma exosome fraction. The whole process from plasma pretreatment to exosome fraction obtaining does not exceed two hours. Characterization of immunoblotting proves that the removal rate of high-density lipoprotein (HDL) can reach 95%, and the removal rate of low-density lipoprotein or very low-density lipoprotein (VHDL) can reach 85%.

Description

technical field [0001] The invention belongs to the technical field of liquid chromatography separation, and in particular relates to a separation method of human plasma exosomes. Background technique [0002] Exosomes are small vesicles secreted by cells, mostly between 30-150 nm in size, have a phospholipid bilayer structure, and contain biomolecules such as DNA, RNA, and protein. Exosomes transport these important biomolecules to recipient cells and can mediate intercellular signal communication. Exosomes play an extremely important role in biological processes such as cell proliferation, immune response, and tissue repair. Exosomes derived from tumor cells often carry cancer-related biomarkers, making them have great potential in clinical cancer diagnosis. More importantly, exosomes appear in various biological fluids, such as blood, urine, saliva, etc., so samples can be collected and purified in a non-invasive way. [0003] Among many biological fluids, plasma is th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/74
CPCG01N30/02G01N30/06G01N30/74G01N2030/027
Inventor 高明霞郑浩洋王轩堂严少涵张祥民
Owner FUDAN UNIV
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