Magnetic Bead Based Proximity Extension Assay for Sensitive Protein and Extracellular Vesicles Detection

During the early stages of disease development, protein biomarkers can leak into the blood creating opportunities for early diagnosis of disease with minimally invasive sampling. These proteins biomarkers however are often masked by the presence of more abundant functional blood proteins, making specific detection a challenge with most current immunoassays. We we report on the development of a magnetic bead based solid-phase PEA (SP-PEA) for sensitive detection of proteins in plasma and serum samples. Antibody functionalized magnetic beads are used to capture the target of interest. Following capture, non-specifically bound proteins are washed off before PEA probes are added for detection of the bound proteins. Compared to hoogenous PEA, SP-PEA admits the use of larger sample volumes to increase available target molecules, higher concentration of detection reagents for more efficient formation of detection complexes and washes for removal of nonspecific background. We compared SP-PEA to solution phase PEA for the detection of cytokines: interlukin-6, interlukin-2, interlukin-4, interlukin-10 and Tumor Necrosis Factor-alpha, and we demonstrated an increased sensitivity by 15 to 60 fold in buffer and chicken serum. We further expanded SP-PEA to detect extracellular vesicles (EVs) through combinations of proteins on the surface of specific EV populations.