Fast isolation of highly specific population of platelet-derived extracellular vesicles from blood plasma by affinity monolithic column, immobilized with anti-human CD61 antibody

Abstract

A new, fast and selective immunoaffinity chromatographic method including a methacrylate-based convective interaction media (CIM ®) disk monolithic column, immobilized with anti-human CD61 antibody, was developed for the isolation of CD61-containing platelet-derived extracellular vesicles (EVs) from plasma. The isolated EVs were detected and size characterized by asymmetrical flow field-flow fractionation (AsFlFFF) with multi-angle light-scattering (MALS) and dynamic lightscattering (DLS) detection, and further confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The mean size of platelet-derived EVs isolates from the anti-CD61 CIM ® disk monolithic column were 174 nm (SD 60 nm) based on the NTA results. These results indicated a successful isolation of platelet-derived EVs, which was confirmed by Western blotting the isolates against the EV-specific markers CD9 and TSG101 together with transmission electron microscopy. Additional elucidation of MALS and DLS data provided detailed information of the size distribution of the isolated fractions, confirming the successful isolation of also small platelet-derived EVs ranging from 30 to 130 nm based on the hydrodynamic radii. The isolation procedure took only 19 min and the time can be even further decreased by increasing the flow rate. 2 The same immunoaffinity chromatographic procedure, following AsFlFFF allowed also the isolation and characterization of platelet-derived EVs from plasma in under 60 minutes. Since it is possible to regenerate the anti-CD61 disk for multiple uses, the methodology developed in this study provides a viable substitution and addition to the conventional EV isolation procedures.

Changes in the proteome of extracellular vesicles shed by rat liver after subtoxic exposure to acetaminophen

“…To circumvent this problem, a membrane containing vesicle fraction was isolated using immobilized monoclonal antibodies against antigens known to be exposed on the cell surface [53]. Following affinity chromatographic enrichment, different sample prepara-tion methods, like gel-in-tube tryptic digestion, were employed [35,36], a strategy has been used for the isolation of exosomes [32,54]. In the step of gel-in-tube digestion, EVs were incorporated into a polyacrylamide gel matrix and subsequently digested with trypsin.…”

Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo—Implications for biomarker discovery

“…The use of dUC is another limitation of this study, because dUC is known to co-isolate protein aggregates and lipoproteins [44,45]. Additionally, the recovery of EVs with dUC is variable (10-80%) and often low [46] and it excludes the smallest of EVs and exomeres, to which little attention has been focused so far [47,48]. Thus, this study did not address the possible alterations in the smallest EV subpopulations.…”

Advances in Exosomes‐Based Drug Delivery Systems

“…Finally antibodies linked to a column allow for exosomes separation by immunoaffinity chromatography. [ 92 ]…”

“…To implement them, it is necessary to identify common exosome surface components, such as the membrane protein trespanin CD63, and immobilize the corresponding antibodies in an adequate platform. [92,93] Magnetic microbeads facilitate the isolation via magnetoimmnunoaffinity. [94] Furthermore, the use of defined antibodies allows for the isolation of specific exosomes subpopulations.…”

“…[98] Finally antibodies linked to a column allow for exosomes separation by immunoaffinity chromatography. [92] Recently, microfluidic devices are used in exosome isolation [99,100] (Figure 5). Their basis is the controlled handling of the sample through capillary tubes associated to different isolation mechanisms.…”