TITLE: EVTrap non-antibody affinity bead protocol for EV isolation from urine, compatible with SiMOA assays
AUTHORS: andrew.west, yuan.yuan

[DESCRIPTION]
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.

[STEPS]
SECTION: Preparation of Urine
1.     Prechill the AVANTI centrifuge, add adapters (Prechilled).
 

SECTION: Preparation of Urine
2. Thaw the urine in a shaking water bath at 42 °C 100 rpm . Leave the urine tubes on ice.
 


SECTION: Preparation of Urine
3. Aliquot 250uL of urine into labeled 1.5mL tubes for creatinine measurements.
SECTION: Preparation of Urine
4. Transfer 5mL of urine to labeled 15mL conical tubes (keep tubes on ice )

SECTION: Preparation of Urine
5. Add ddH2O to each urine sample tube until volume reaches 7mL (keep on ice )

SECTION: Preparation of Urine
6. Centrifuge urine at 10000 x g  for 10 min at4 °C to remove debris and large apoptotic bodies.

SECTION: Preparation of Urine
7. Collect the cleared urine (keep on ice ), leaving the pellet behind (keep on ice ). 

SECTION: Capture of Extracellular Vesicles
8. Add the provided 10x Urine/Media Loading Buffer at 1-to-10 ratio to the urine sample (e.g. 700uL to 7mL urine). 
SECTION: Capture of Extracellular Vesicles
9. Add 60uL Tymora EVtrap beads reagent. 
SECTION: Capture of Extracellular Vesicles
10. Incubate for 240 min by end-over-end rotation at 4 °C  (make sure the beads freely move around). 

SECTION: Capture of Extracellular Vesicles
11. Spin samples at low speed, 200 x g (but, use 500x g if you don’t see the right compact pellet) for 5 min. Check for compact pellet at bottom of tube.

SECTION: Capture of Extracellular Vesicles
12. 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).
SECTION: Capture of Extracellular Vesicles
13. Remove supernatant and transfer to temporary tube. 
SECTION: Capture of Extracellular Vesicles
14. 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. 
SECTION: Capture of Extracellular Vesicles
15. Transfer supernatant back to original 15mL tube and save in -80 °C  .

SECTION: Capture of Extracellular Vesicles
16. Isolate beads using magnetic separator and remove the wash solution. 
SECTION: Capture of Extracellular Vesicles
17. Repeat wash step with cold PBS two more times, 1 mL each wash. Invert 10 times each. 
SECTION: Capture of Extracellular Vesicles
18. Pulse spin (to get liquid out of cap). 
SECTION: Capture of Extracellular Vesicles
19. 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 10 min by vigorous shaking at 1000 rpm (eppemixer) at Room temperature  . 

SECTION: Capture of Extracellular Vesicles
20. Place tube on a magnetic separator and collect elution buffer (~50ul) (this contains the EVs). 
SECTION: Capture of Extracellular Vesicles
21. Repeat the elution step one more time with another 50uL of Tymora Elution solution and combine both elutions together. (~100uL) 
SECTION: Capture of Extracellular Vesicles
22. Adjust pH to 7.4 using 1N HCl (make fresh, very carefully!) (7uL of acid per 100uL of elution solution).
SECTION: Capture of Extracellular Vesicles
23. 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). 
SECTION: Capture of Extracellular Vesicles
24. 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).
SECTION: Capture of Extracellular Vesicles
25. Aliquot half of sample to a labeled 1.5mL Protein Low-Binding tube and store at -80 °C for future use.