Development of Multiplexed Bead-Based Immunoassays for Profiling Soluble Cytokines and CD163 Using Mass Cytometry

[Image: see text] Bead-based immunoassays are multiparametric analysis allowing for the simultaneous quantification of a large number of biomarkers within a single sample. Mass cytometry is an emerging cytometric technique that offers a high multiplexing capacity in a high-throughput setting but has not yet been applied to bead-based assays. In this study, we developed a multiplex bead-based immunoassay of cytokines and CD163 designed for mass cytometry (MC). A set of 11 types of lanthanide-encoded microbeads were synthesized by two-stage dispersion polymerization as classifier candidates for the assay. These beads were then decorated with different Abs on the surface to capture the target cytokines in solution. Gold nanoparticles were employed as reporters to identify the binding of target cytokines on the classifier surface. As a proof-of-concept study, we first developed four-plex and nine-plex assays of mixtures of cytokines in standard solutions. The MC signal intensities of these immunoassays were responsive to the concentration differences in the standard solutions with high detection sensitivities at low analyte concentrations. Finally, we examined a sample of peripheral blood mononuclear cells (PBMCs) with the nine-plex assay, comparing an unstimulated sample with a sample stimulated to promote cytokine secretion.