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Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research.

Scientific reports (2017-06-04)
Glenn Vergauwen, Bert Dhondt, Jan Van Deun, Eva De Smedt, Geert Berx, Evy Timmerman, Kris Gevaert, Ilkka Miinalainen, Véronique Cocquyt, Geert Braems, Rudy Van den Broecke, Hannelore Denys, Olivier De Wever, An Hendrix
ABSTRACT

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Green Fluorescent Protein Antibody, Chemicon®, from mouse
Sigma-Aldrich
Anti-PMP70 antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Millipore
Amicon® Ultra-2 Centrifugal Filter Unit, Ultracel-10 regenerated cellulose membrane, 2 mL sample volume