Automation and High Throughput Technologies
While antibody-based assays such as sandwich ELISA and AlphaLISA can detect unmodified proteins for high-throughput screening (HTS), they have constraints due to the quality and availability of two primary antibodies, excessive costs, and technical considerations. An alternative is the reverse phase protein array (RPPA) in which nanoliter spots of cell lysate are arranged in high density onto a protein binding substrate, typically nitrocellulose-coated glass slides using tip-based arrayers, to enable endogenous protein quantification by ubiquitous immunochemical protocols. Although RPPA only requires a single antibody, instrumentation compatibility and costs associated with coated slides can be prohibitive for large-scale HTS.
To address this, we first designed a cell-based 1536-well HTS assay utilizing acoustic droplet ejection to sample nanoliter volumes of media and quantify a secreted bioluminescent reporter. We then adapted this technique for RPPA as an orthogonal antibody-based methodology by constructing a 3D-printed, low-cost nitrocellulose membrane plate alternative to coated slides. In parallel seven-concentration LOPAC1280 qHTS experiments, consistent performance between assays orthogonally identified secretory modulators of the reporter. This acoustically arrayed immunoassay, referred to as acoustic RPPA, enabled quantification of native, endogenously secreted protein with picogram sensitivity and multiplexing capabilities with cytotoxicity and imaging assays. The acoustic RPPA methodology can therefore be utilized as a powerful standalone immunochemical technique or component of a robust drug discovery platform that generates extensive biological profiles from individual wells using physiologically relevant cellular systems, as demonstrated with iPSC-derived hepatocytes. Next generation acoustic RPPA plates, designed to be more automation and reader friendly, have been manufactured and are currently under evaluation alongside custom machined HTS labware.