Kit and method for preparing exosomes in serum or plasma

A technology of exosomes and kits, applied in the field of biomedicine, can solve the problems of large particle range, low yield, and low purity, and achieve the effects of concentrated particle size distribution, reduced interference, and guaranteed activity

Active Publication Date: 2018-11-30
BEIJING TRANSGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the exosomes extracted by the methods of the prior art have a low yield, a large particle range, and low purity, and tend to contain protein particles similar in size to the exosomes

Method used

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  • Kit and method for preparing exosomes in serum or plasma
  • Kit and method for preparing exosomes in serum or plasma
  • Kit and method for preparing exosomes in serum or plasma

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preparation example Construction

[0069] In addition, the present invention also provides a method for preparing exosomes in serum or plasma, comprising the following steps:

[0070] Add any of the above-mentioned exosome extraction reagents to the serum or plasma sample, mix well, let stand, and centrifuge to remove the supernatant to obtain exosome precipitation; the amount of exosome extraction reagent is 1 / 2 of the volume of the sample to be extracted. 1 / 4 times - 1 times, preferably 1 / 4 times - 1 / 2 times; the condition of standing still is preferably 4 ℃ for 10-60 minutes, more preferably 20-40 minutes, when sufficient settlement is ensured At the same time, the activity of exosomes is guaranteed; the centrifugation condition is preferably 2000g-15000g for 10 minutes, more preferably 8000g-12000g for 10 minutes.

[0071] Add the above-mentioned exosome lysis reagent to the exosome precipitation to obtain an exosome lysis solution; the exosome lysis reagent is preferably PBS buffer solution.

[0072] Add ...

Embodiment 1

[0087] Extraction of exosomes from serum

[0088] The composition of the reagents used:

[0089] Extraction reagent P1 (dextran sulfate 13,000, 5%, glucose, 2.5%)

[0090] Dissolving reagent S1 (PBS solution)

[0091] Purified Microspheres B1 (Protein G and Butyl-Sepharose 4B, 20:1)

[0092] 1. Centrifuge at 3,000g 4°C for 15 minutes to remove residual cells and debris in the serum.

[0093] 2. Add 25 μl P1 to 100 μl serum for each sample, mix by inversion, let stand at 4°C for 30 minutes, and centrifuge at 10,000 g for 10 minutes.

[0094] 3. Discard the supernatant, collect the precipitate, centrifuge again at 10,000g for 5 minutes, and discard the remaining supernatant.

[0095] 4. Add the S1 solution twice the initial volume, and gently pipette to dissolve to obtain the exosome solution.

[0096] 5. Take out purified microspheres B1 equal in volume to the initial plasma volume, centrifuge at 4,000g for 3 minutes, discard the supernatant, add 1ml of S1 solution and mix...

Embodiment 2

[0100] Extraction of exosomes from plasma

[0101] The composition of the reagents used:

[0102] Extraction Reagent P1 (PEG 30,000, 5%, Dextran Sulfate 15,000, 5%, Glucose, 6%)

[0103]Dissolving reagent S1 (PBS solution)

[0104] Purification Microspheres B1 (Protein G and Butyl-Sepharose 4B, 1:1)

[0105] Proteinase K (20%)

[0106] 1. Centrifuge at 3,000g 4°C for 15 minutes to remove residual cells and debris in the serum.

[0107] 2. Transfer the supernatant to a new EP tube, add 0.5 times the volume of S1 solution, invert or vortex to mix.

[0108] 3. Add 0.05 times the volume of Proteinase K, mix the sample, incubate at 37°C for 10 minutes, and then pre-cool to 4°C.

[0109] 4. Add 0.2 times the volume P1 of the existing total volume (volume of plasma + volume of S1) to the sample, mix well and place at 4°C for 30 minutes.

[0110] 5. Centrifuge at 10,000 g at 4°C for 10 minutes.

[0111] 6. Collect the precipitate, discard the supernatant, centrifuge again at 10...

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Abstract

The invention discloses a kit and method for preparing exosomes in serum or plasma. The kit comprises an exosome extracting reagent, wherein the exosome extracting reagent is a hydrophilic polymer ora solution of the hydrophilic polymer; the hydrophilic polymer comprises one or a combination of PEG (Poly Ethylene Glycol), dextran sulfate and PVP (Poly Vinyl Pyrrolidone); the molecular weight of the PEG is 20000-1200000 Daltons; the molecular weight of the dextran sulfate is 5000 to 20000 Daltons; and the molecular weight of the PVP is 10000 or 40000-700000 Daltons. The particle size distribution of the exosomes obtained by utilizing the kit is more concentrated; and according to the method for preparing the exosomes, which is provided by the invention, the step of purifying the exosomes is also optimized, so that the exosomes have less impurities and higher purity.

Description

technical field [0001] The invention relates to the technical field of biomedicine. More specifically, it relates to a kit for preparing exosomes in serum or plasma and a method for preparing exosomes. Background technique [0002] Discovered in 1986, exosomes are double-layer lipid membrane vesicles with a diameter of about 30-150nm, which can be produced by various cells in the body such as immune cells, stem cells, cardiovascular cells, and reticulocytes. , platelets, nerve cells and tumor cells are actively secreted and widely distributed in peripheral blood, urine, saliva, milk, ascites, amniotic fluid and other body fluids. Exosomes carry a large number of specific proteins (such as cytokines, growth factors) and functional mRNA, miRNA and other biologically active substances. At present, people usually use methods such as ultracentrifugation, immunomagnetic beads, ultrafiltration, precipitation or kits to achieve the extraction and separation of exosomes. However, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
CPCG01N1/28
Inventor 李晶曹洋马静辛文
Owner BEIJING TRANSGEN BIOTECH CO LTD
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