An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization
Fig 2
Presence of plasma proteins leads to co-isolation of etanercept in the SEC pEV fraction.
Etanercept (1μg/ml) in PBS alone or with pfp was SEC isolated. In each eluted SEC fraction, TNF-α inhibition was measured in triplo (A). Etanercept (1μg/ml) in the presence of 3 different concentrations pfp (10-20-100%) or equal protein levels of albumin (7-21-70 mg/ml) was SEC isolated. In Fraction 5 (pEV fraction) TNF-α inhibition was measured in triplo (B). Next, levels of etanercept were measured in plasma of 6 new etanercept treated RA patients (C). Finally, in these RA patients neutralization of TNF-α was measured in triplo on SEC isolated pEVs before (white bars) and after (black bars) etanercept treatment (C). Statistically significant differences in TNF-α reduction versus control (only TNF-α without pEVs), were determined by Mann-Whitney test, * p<0.05, ** p<0.01, *** p<0.001.