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Multi-column for isolating exosomes and exosome isolation method

A separation method and technology of exosomes, applied in separation methods, solid adsorbent liquid separation, peptide preparation methods, etc., can solve problems such as long time, loss of exosomes, and in-depth analysis of lipoprotein elution. , to achieve the effect of improving purity and excellent separation efficiency

Active Publication Date: 2020-10-13
KOREA UNIV RES & BUSINESS FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, when using existing column chromatography to separate exosomes, there are problems in that lipoproteins (lipoprotein) are eluted together with exosomes, or that exosomes are lost when more than two separation steps are performed or takes a long time
Existing column chromatography studies mainly focus on the exosome elution section, with little in-depth analysis of lipoprotein elution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: the preparation of multicolumn

[0065] Sepharose (Sepharose) CL-6B was filled with 70% (v / v) in the lower part (diameter: 10mm, height: 50mm) of the column used for chromatography, and acryl-dextran gel (Sephacryl) 200 - HR was filled in its upper part at 30% (v / v), so that a multi-column was prepared so that the height ratio of the lower part and the upper part was 7:3. Here, the average surface charge values ​​of Sepharose CL-6B and Sephadex 200-HR were -30 and -7, respectively.

Embodiment 2

[0066] Example 2: Confirmation of Exosome Separation Ability from Existing Column Conditions

[0067] A column for chromatography packed with beads (diameter: 10 mm, height: 50 mm) was used, and as a sample, supernatant serum produced after centrifugation at 10000 rcf at 4° C. for 30 minutes was used. Size Exclusion Chromatography (SEC, Size Exclusion Chromatography) was performed on the samples in order to determine the separation efficiency of the material by existing single and multi-columns.

[0068] Specifically, as the beads packed in the column, Sepharose CL-2B was used as a single column (2B), and (Dual column) prepared in Example 1 was used as a multi-column. Beads filled with 300 ml PBS were washed before loading samples and the prepared samples were loaded just in time when the mobile phase buffer (except packed column beads) was exhausted. The volume of sample loaded on the column should not exceed 5% of the total volume of the packed beads, and 0.3 to 0.5 ml of...

Embodiment 3

[0070] Example 3: Comparison of the elution trends of exosomes and lipoproteins in serum

[0071] In order to compare the elution of exosomes, lipoproteins and water-soluble proteins in each segment in serum samples, the multi-column of Example 1 was used to fractionate by the method described in Example 2, and each Fractions were subjected to SDS-PAGE and Western blot. As the primary antibody, an anti-CD63 antibody (sc-15363 manufactured by Santa Cruz) as an exosome marker protein was diluted at 1:500, and an ApoB-100 antibody (sc-15363 manufactured by Santa Cruz) as a lipoprotein antibody was used. 25542) was diluted 1:1000, and then the diluted antibody was used to confirm its elution.

[0072] As a result, albumin (˜65 kDa) was detected as a water-soluble protein after the 13th segment ( FIG. 3 ), and lipoproteins appeared most prominently in the 9th segment and were then detected until the 11th segment. After a small amount of exosomes were detected in the 9th segment...

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Abstract

The present invention relates to a multi-column for isolating exosomes and an exosome isolation method, for isolating exosomes from a biological sample containing exosomes mixed with impurities such as lipoproteins and water-soluble proteins.

Description

technical field [0001] The invention relates to a multi-column and exosome separation method for high-purity separation of exosomes from a biological sample in which various biomolecules and exosomes are mixed. Background technique [0002] Extracellular vesicles include exosomes, ectosomes, microvesicles, and apoptotic bodies released or secreted by cells. The size is 20-150nm, and it is a biological nanoparticle produced by multivesicular bodies (MVB, Multivesicular bodies) in cells. [0003] These extracellular vesicles can be relatively easily isolated from various biological fluids (biofluid) such as blood, lymph fluid, cerebrospinal fluid, urine, amniotic fluid, breast milk, saliva, semen, etc., and are called dexosomes ( dendritic cells), oncosomes (derived from cancer cells), prostasomes (derived from prostate cells), cardiosomes (derived from cardiomyocytes), and the like. Extracellular vesicles contain various nucleotides or tagged proteins depending on their cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16B01D15/22
CPCB01D15/22B01D15/1871B01D15/34G01N30/6039B01J20/28083B01J20/28085B01J20/28052B01J20/283B01J20/285B01J2220/54G01N2030/8831G01N30/80G01N2030/027
Inventor 朴龙崔然皓洪性会金贤九朴志镐郑直汉
Owner KOREA UNIV RES & BUSINESS FOUND
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