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

Abstract

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.

Recent Advances in Surface Plasmon Resonance (SPR) Technology for Detecting Ovarian Cancer Biomarkers

“…Some biomarkers incorporated in multivariate algorithms for EOC may lack sufficient specificity to reliably detect the disease. Moreover, the lack of S P these biomarkers to a particular tumor or tissue type, along with their presence in non-cancerous conditions, limits their suitability for screening and early-stage diagnosis [43,45,[56][57][58]. Additionally, the widely used single detection biomarkers, CA125 and HE4, employ ECLIA and magnetic-bead assay detection methods.…”

“…An emerging trend in immunoassays is the development of solid support versions utilizing microbeads, particularly magnetic beads with paramagnetic properties. These micrometer-sized magnetic beads allow for the removal of non-specifically bound serumbased biomarkers and are embedded in a non-magnetic/polymer matrix [57,58,71]. Examples of magnetic bead-based assays include the Luminex multiplex assay (Microplex, Microspheres, MagPlex) utilizing Luminex xMAP technology and Proseek plates employing Proximity Extension Assay (PEA) technology for biomarker detection.…”

“…The PEA platform (Olink Proteomics AB, Uppsala, Sweden), as presented, offers the capability to simultaneously detect 92 proteins in 96 samples, creating possibilities for large-scale generation of protein biomarker data [58]. PEA technology excels in three crucial immunoassay parameters: high SN%, high SP%, and low sample consumption.…”