Exosomes Derived Neuronal Markers: Immunoaffinity Isolation and Characterization

AbstractNeuronal exosomes play a crucial role in intercellular communication in the brain and represent a promising biomarker for neurological diseases, including stroke. However, limited techniques are available for isolating neuronal exosomes due to their small number in the serum exosomes. Thus, the development of efficient tools with brain-specific markers is needed. Here, we show the optimization of an immunoaffinity assay-based isolation protocol for specific exosomes or neuronally derived exosomes (NDE). Our results demonstrated that one-micron functionalized magnetic beads successfully separated CD63+ and L1CAM+ exosomes from serum. The size and shape of exosomes or exosomes pulled by beads were confirmed by Dynamic light scattering and Transmission electron microscopy; also, beads were well resolved in conventional flow cytometry analysis, which revealed that CD63-pulled serum exosomes had 5% expression of L1CAM. Furthermore, transmission electron microscopy showed that exosomes eluted from magnetic beads retained their original size, shape, and form without any damage. Furthermore, we showed isolation of NDE using GluR2/3-capturing antibody ( α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor) using an optimized immunoaffinity bead assay utilizing 100 µl serum of stroke patients or age-matched healthy group. GluR2/3-captured exosomes were confirmed by western blot analysis. The western blot analysis showed a signifi cant increase in the 35KDa s...

Source: NeuroMolecular Medicine - November 22, 2021 Category: Neurology Source Type: research