*2.4.2 Biochemical analysis*

The characterization of EVs to determine the surface markers, markers of origin and proteins they carry allows to infer the functional role of these vesicles in health and disease. Methods might be divided to more conventional ones; the immunoblotting assays or the methods that will employ the capture of the vesicle; immunosorbent methods. Immunoblotting methods are based on the lysis of a vesicle and the analysis of its contents either by direct spotting on a membrane (dot blot) or separation of proteins using SDS PAGE combined with western blotting, in which specific proteins of interest are determined with labeled antibodies. Immunoblotting methods are often used to determine the presence of EVs in a sample. These methods can also be used to determine the purity of samples [18]. Immunosorbent assays are based on the detection of EVs using specific antibodies directed against surface proteins of EVs. Derived from the classical enzyme linked immunosorbent protein assay (ELISA), EVs are captured on a solid surface coated with antibodies that are typically present on the EVs. EVs capture results in a

strong enrichment. Analysis of EVs surface proteins is afterwards performed with antibodies directed to a protein of interest on the surface of the EVs. These detection antibodies are conjugated to an enzyme enabling the conversion of a fluorescent/ colored substrate that can be quantified with a spectrophotometer [18].
