Jan 19, 2024

Public workspaceEVTrap non-antibody affinity bead protocol for EV isolation from urine, compatible with SiMOA assays

DOI
dx.doi.org/10.17504/protocols.io.81wgbxonylpk/v1
  • 1Duke University
andrew.west west
Open access
DOI: dx.doi.org/10.17504/protocols.io.81wgbxonylpk/v1
Protocol Citation: andrew.west west, yuan.yuan 2024. EVTrap non-antibody affinity bead protocol for EV isolation from urine, compatible with SiMOA assays. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxonylpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 04, 2024
Last Modified: January 19, 2024
Protocol Integer ID: 92957
Keywords: ASAPCRN
Funders Acknowledgement:
NIH/NINDS
Grant ID: NS064934
Michael J. Fox Foundation for Parkinson’s Disease Research
Grant ID: MJFF-022434
Abstract
A modified protocol using EVTrap beads (Tymora Analytical) to isolate EVs from human urine, and subsequent processing, for detection of proteins using SiMOA or comparable ELISA-based protocols.
Materials
Reagents:
  1. Tymora 10x Loading Buffer
  2. Tymora Elution Buffer
  3. Tymora EVTrap Beads

Preparation of Urine
Preparation of Urine
Prechill the AVANTI centrifuge, add adapters (Prechilled).
Note
Do a pre-run with 10 of 15 mL conical tubes with water, to check for breakage or plastic warping), run for Duration00:20:00 at Centrifigation1000 x g , Temperature4 °C .


Thaw the urine in a shaking water bath at Temperature42 °C Centrifigation100 rpm . Leave the urine tubes TemperatureOn ice .
Note
Do not thaw at room temperature or in a refrigerated environment


Aliquot 250uL of urine into labeled 1.5mL tubes for creatinine measurements.
Transfer 5mL of urine to labeled 15mL conical tubes (keep tubes TemperatureOn ice )

Add ddH2O to each urine sample tube until volume reaches 7mL (keep TemperatureOn ice )

Centrifuge urine at Centrifigation10000 x g for Duration00:10:00 atTemperature4 °C to remove debris and large apoptotic bodies.

10m
Collect the cleared urine (keep TemperatureOn ice ), leaving the pellet behind (keep TemperatureOn ice ).

Capture of Extracellular Vesicles
Capture of Extracellular Vesicles
4h 15m
Add the provided 10x Urine/Media Loading Buffer at 1-to-10 ratio to the urine sample (e.g. 700uL to 7mL urine).
Add 60uL Tymora EVtrap beads reagent.
Incubate for Duration04:00:00 by end-over-end rotation at Temperature4 °C (make sure the beads freely move around).

4h
Spin samples at low speed, Centrifigation200 x g (but, use 500x g if you don’t see the right compact pellet) for Duration00:05:00 . Check for compact pellet at bottom of tube.

5m
Prepare the washing buffer by diluting the 10x Urine/Media Loading Buffer at 1-to-10 ratio in PBS (e.g. 2mL buffer to 20mL PBS).
Remove supernatant and transfer to temporary tube.
Transfer beads to a labeled 1.5mL tube in 1mL of wash buffer (1x washing buffer) by inverting the beads 10-20 times and removing the wash solution.
Transfer supernatant back to original 15mL tube and save in Temperature-80 °C .

Isolate beads using magnetic separator and remove the wash solution.
Repeat wash step with cold PBS two more times, 1 mL each wash. Invert 10 times each.
Pulse spin (to get liquid out of cap).
Add 50 μL of the fresh Tymora Elution solution to the beads (the elution volume should be enough to fully resuspend the beads and allow for efficient interaction) and incubate beads Duration00:10:00 by vigorous shaking at Centrifigation1000 rpm (eppemixer) at TemperatureRoom temperature .

10m
Place tube on a magnetic separator and collect elution buffer (~50ul) (this contains the EVs).
Repeat the elution step one more time with another 50uL of Tymora Elution solution and combine both elutions together. (~100uL)
Adjust pH to 7.4 using 1N HCl (make fresh, very carefully!) (7uL of acid per 100uL of elution solution).
Add 200 uL of Quanterix Homebrew sample diluent buffer (supplemented with 0.1% sodium dodecyl sulfate, 0.05% sodium deoxycholate, and 0.4 mM freshly prepared dithiothreitol).
If the sample is ice, sonicate sample 10sec at 10% power straight. >5 min wait on ice. 10sec at 10% power straight. (~20 sec of straight 10% probe-tip sonication).
Aliquot half of sample to a labeled 1.5mL Protein Low-Binding tube and store at Temperature-80 °C for future use.