Proximity Extension Assay : a universal tool for proteomics in precision medicine

Precision medicine aims to utilise biomarkers to ensure the right treatment is given to the right patient at the right time. In pursuit of this goal, sensitive protein detection is essential to improve opportunities for discovery, verification, validation and development of valuable protein biomarkers. The proximity Extension Assay (PEA) is a commercially established multiplex immunoassay that combines the specificity of dual antibody recognition assays with qPCR sensitivity to parallel analysis of up to 1161 human proteins in a single microlitre of plasma. Already widely applied in biomarker research, in this thesis we report on further developments of the PEA assay format, as well as applications for analysing disease responses and drug-protein engagement. In paper I, we describe the development of a magnetic bead-based solid phase proximity extension assay (SP-PEA) for improved protein and exosome detection. We demonstrate increased sensitivity by up to two orders of magnitude for the detection of cytokines ( IL-8, TNF-alpha, IL-10 and IL-6) as compared to the corresponding homogenous PEA assays. The method is also used for detection of exosomes in plasma samples.In paper II, we have adapted the PEA assay for detection of anti-SARS-CoV-2 antibodies in blood samples. We established the Antibody Proximity Extension Assay (Ab-PEA) as a sensitive and accurate method for detecting anti-SARS-CoV-2 antibodies, serving as a high-throughput solution for community surveillance of virus exposure and/or for testing the efficiency of vaccine induced immunity. In paper III, we use PEA to measure binding by small drug molecules to specific on and off-target proteins via cellular thermal shift assays (CETSA). The approach allows for analysis of target engagement by drugs in minimal samples of cells and tissues, with a potential for high multiplexing and high-throughput in clinically relevant samples. In paper IV, finally, we explore the use of recombinant single-chain binders (nanobodies) as substitutes or complements for antibodies in PEA. We further demonstrate the potential of PEA as a method to identify suitable pairs of protein binders for use in dual binder assays