Fig 2: Affinity proteomics technologies. (A) Suspension bead array assay (Luminex): All proteins within the plasma sample are labelled with biotin for subsequent detection (A1) before incubation with a suspension of multiplexed protein-specific antibodies, each coupled to a unique color-coded micrometer-sized bead (A2). Unbound proteins are removed by washing and fluorescent-labeled-streptavidin is added to detect the protein biotin tag (A3). The suspension is analyzed by a cytometry-based instrument (Luminex), where the color of the antibody-bound bead provides the antibody identification, and the mean fluorescence intensity provides a relative measure of the corresponding target protein levels. (B) Proximity extension assay (Olink): for each biomarker, a matched pair of antibodies recognizing neighboring epitopes on the same target protein are linked to unique complementary oligonucleotides. Multiplex panels of matched antibody pairs are added to plasma (B1). When binding in close proximity on the protein target, the stretch of nucleotides hybridizes. DNA polymerase is added, and the annealing product is extended and amplified (B2) and detected by either quantitative polymerase chain reaction or next-generation sequencing (B3). The readout provides a relative measure of respective protein level in plasma. (C) SomaScan Assay (SomaLogic): for each biomarker, a single-stranded oligonucleotide, aptamer, folded into a tertiary structure, binds the target protein with affinity and specificity comparable to antibodies. The SomaScan platform is based on slow off-rate modified DNA aptamers (SOMAmers) labeled with a fluorescence reporter, photocleavage linker, and biotin. Multiplex panels of SOMAmers together with streptavidin coated beads bind respective target proteins in plasma (C1). Following washing to remove unbound proteins, fluorescent-labeled aptamer-protein complexes are released from the beads by ultraviolet-induced photocleavage, labeled with biotin (C2) and captured on fresh streptavidin-coated beads added to the sample, followed by washing to remove nonspecifically bound aptamers (C3). Protein-bound aptamers are released in denaturing buffer and detected by hybridization to complementary oligonucleotide probes on a microarray chip with the fluorescence intensity (C4) providing a relative measure of respective protein level in plasma. Figure created with BioRender.com