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

Bead-based immunoassaysare multiparametric analysis allowing forthe simultaneous quantification of a large number of biomarkers withina single sample. Mass cytometry is an emerging cytometric techniquethat offers a high multiplexing capacity in a high-throughput settingbut has not yet been applied to bead-based assays. In this study,we developed a multiplex bead-based immunoassay of cytokines and CD163designed for mass cytometry (MC). A set of 11 types of lanthanide-encodedmicrobeads were synthesized by two-stage dispersion polymerizationas classifier candidates for the assay. These beads were then decoratedwith different Abs on the surface to capture the target cytokinesin solution. Gold nanoparticles were employed as reporters to identifythe binding of target cytokines on the classifier surface. As a proof-of-conceptstudy, we first developed four-plex and nine-plex assays of mixturesof cytokines in standard solutions. The MC signal intensities of theseimmunoassays were responsive to the concentration differences in thestandard solutions with high detection sensitivities at low analyteconcentrations. Finally, we examined a sample of peripheral bloodmononuclear cells (PBMCs) with the nine-plex assay, comparing an unstimulatedsample with a sample stimulated to promote cytokine secretion.