Antibody-Based Assays for Phenotyping of Extracellular Vesicles

Figure 7

Schematic view of the bead-based fluidic assay. For this microfluidic assay, a prototype polydimethylsiloxane (PDMS) chip was developed. The device is designed with four inlets and a cascading microchannel circuit for sequential isolation and phenotyping of EVs. Plasma is premixed with antibody-labeled magnetic beads and introduced onto a PDMS chip, where bead-labeled EVs are magnetically retained. The EVs are subsequently lysed and the released proteins (surface and intravesicular antigens) are captured by antibody-labeled magnetic beads and detected by a sequential introduction of the primary detection antibody, the secondary antibody (alkaline phosphatase labeled), and a fluorogenic substrate (DiFMUP) for a sandwich immunodetection of the antigens of interest. An epifluorescence microscope equipped with a mechanical shutter and a CCD camera was used for chemifluorescence detection. A filter set (excitation 325–375, emission 435–485) was employed for detection. By the use of protein standards, the assay provides a quantitative detection of the protein of interest.