Method for detecting extracellular vesicles in a sample

Patent metadata

Field	Value
Publication number	US-2020200740-A1
Application number	US-201816612773-A
Country	US
Kind code	A1
Filing date	May 16, 2018
Priority date	Jun 13, 2017
Publication date	Jun 25, 2020
Grant date	—

Abstract

A method for detecting extracellular vesicles in a sample, including the following steps: (a) applying the sample to a substrate, (b) adding probes suitable for detection which mark the extracellular vesicles by specific binding to them; and (c) detecting the extracellular vesicles by measuring a specific signal from the probes; wherein step (b) can be performed prior to step (a).

First claim

1 . A method for detecting extracellular vesicles in a sample, comprising the following steps:
a) applying the sample to a substrate, b) adding probes suitable for detection which mark the extracellular vesicles by specific binding to them; and c) detecting the extracellular vesicles by measuring a specific signal from the probes, wherein step b) can be performed prior to step a).

2 . The method according to claim 1 ,
wherein prior to step a), capture molecules for the extracellular vesicles are immobilized on the substrate.

3 . The method according to claim 2 ,
wherein by contacting the capture molecules, the extracellular vesicles are immobilized on the substrate by binding to the capture molecules.

4 . The method according to claim 1 ,
wherein after the extracellular vesicles have contacted the probes, molecules and particles that are not specifically bound are removed by washing.

5 . The method according to claim 1 ,
wherein probes are selected which bind to the extracellular vesicles, wherein the probes are also capable of emitting a specific signal.

6 . The method according to claim 2 ,
wherein the extracellular vesicles contact the capture molecules and the probes simultaneously.

7 . The method according to claim 2 ,
wherein the extracellular vesicles contact the probes prior to contacting the capture molecules.

8 . The method according to claim 1 ,
wherein the sample is fixed before the probes bind to the extracellular vesicles.

9 . The method according to claim 1 ,
wherein the sample is treated with detergents.

10 . The method according to claim 5 ,
wherein a spatially resolved determination of the probe signal takes place.

11 . The method according to claim 1 ,
wherein the substrate comprises plastic, silicon or silicon dioxide.

12 . The method according to claim 1 ,
wherein the substrate has a hydrophilic surface prior to immobilizing capture molecules on the substrate.

13 . The method according to claim 12 ,
wherein the hydrophilic layer is selected from the group comprising or consisting of PEG, poly-lysine, dextran, and derivatives thereof.

14 . The method according to claim 1 ,
wherein a functionalization with amino groups occurs by bringing the substrate into contact with APTES (3-aminopropyl-trietoxy silane) or ethanolamine.

15 . The method according to claim 14 ,
wherein bringing the substrate into contact with APTES (3-aminopropyl-trietoxy silane) occurs in the gas phase.

16 . The method according to claim 2 ,
wherein the capture molecules are covalently bonded to the substrate or to a coating.

17 . The method according to claim 2 ,
wherein binding sites of the extracellular vesicles are epitopes and the capture molecules and probes are antibodies or parts thereof.

18 . The method according to claim 1 ,
wherein the probes are marked with fluorescent dyes.

19 . The method according to claim 1 ,
wherein detection takes place by spatially resolving fluorescence microscopy.

20 . A kit for carrying out the method according to claim 1 , comprising:
a substrate having a hydrophilic surface and/or an immobilized capture molecule; a probe; and solutions and buffers.

Assignees

• Forschungszentrum Juelich Gmbh

Inventors

• Zafiu Christian
• Willbold Dieter
• Kulawik Andreas
• Bannach Oliver

Classifications

• G01N33/5308 Primary

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G01N1/34

  Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)} · CPC title

• G01N33/5076

  involving cell organelles, e.g. Golgi complex, endoplasmic reticulum · CPC title

• G01N33/543

  with an insoluble carrier for immobilising immunochemicals · CPC title