Developing reversible immuno-affinity capture for extracellular vesicles purification

Abstract

Extracellular vesicles (EVs) are a group of cell-secreted supramolecular structures that play a role in important physiological and pathological processes. EVs are present in all bodily fluids. EVs represent unique source of clinically relevant and easily accessible biomarkers. Accordingly, increasing research interests in EVs field is advancing toward their use in precision medicine with particular focus of Liquid Biopsy. Present EVs isolation approaches are very inefficient, time-consuming and expensive. The application of immune-based capture could represent an effective alternative. Further, as an alternative to conventional antibodies, single-domain antibodies (VHH) obtained by direct panning on EVs can enable purification of EVs from different sources (human plasma or conditioned culture medium) with significantly reduced costs 1. The aim of this work was to combine single domain antibody based affinityfor high performance scalable EV capture. In this work, we develop system VHH-methacrylate-based copolymer for purification 2. VHHs used in this work (H1, H6, D5, B1 and G2) were isolated from a naïve pre-immune library by direct panning against EVs from the supernatant of cultured human cells. VHHs cloned with eGFP and 6xHis tag, were produced in E. coli cells, purified and immobilised on polymer and used for immunocapture purification of EVs from tissue culture medium and human plasma. Biochemical and morphological features of the isolated EVs were determined using different methods. Methacrylate-based copolymer was used as a porous solid support, the chemical versatility of which enables its efficient functionalization with VHHs. The combined analyses of morphological features and biomarkers (CD9, CD63 and CD81) presence indicated that the recovered EVs were exosomes. The lipoprotein markers APO-A1 and APO-B were both negative in tested samples. This is the first report demonstrating the successful application of spherical porous methacrylate-based copolymer coupled with VHHs for the exosome isolation from biological fluids. This 67 inexpensive immunoaffinity method has the potential to be applied for the isolation of EVs belonging to different morphological and physiological classes.