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

Prechill the  **AVANTI**  centrifuge, add adapters (Prechilled).

Thaw the urine in a shaking water bath at 42°C 100rpm,0h 0m 0s . Leave the urine tubes On ice.

Aliquot 250uL of urine into labeled 1.5mL tubes for creatinine measurements.

Transfer 5mL of urine to labeled 15mL conical tubes (keep tubes On ice )

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

Centrifuge urine at 10000x g,0h 0m 0s for 0h 10m 0s at4°C to remove debris and large apoptotic bodies.

Collect the cleared urine (keep On ice ), leaving the pellet behind (keep On ice ).

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 4h 0m 0s by end-over-end rotation at 4°C (make sure the beads freely move around).

Spin samples at low speed, 200x g,0h 0m 0s (but, use 500x g if you don’t see the right compact pellet) for 0h 5m 0s. Check for compact pellet at bottom of tube.

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 -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 0h 10m 0s by vigorous shaking at 1000rpm,0h 0m 0s (eppemixer) at Room temperature .

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 -80°C for future use.