Miniaturization of a Lumit p-ERK immunoassay for cell-based high-throughput screening of a chemogenetic...

Cell-based assays that report endogenous signaling events are attractive for high-throughput screening (HTS), but many formats rely on engineered reporters or fluorescence detection that can be limited by background and compound interference. We adapted the bioluminescent Lumit p-ERK1 (T202) immunoassay to 384- and 1536-well plate formats suitable for HTS. Using MCF-7 cells stimulated with EGF or PMA, we optimized lysis conditions and antibody concentrations to maximize signal-to-background while preserving detection of basal p-ERK. In 384-well plates, a manually run screen of a 190-compound FDA-approved Tocris library yielded an average Z' factor of 0.69 and a ∼32-fold signal window and enriched for known MAPK/ERK pathway inhibitors. In 1536-well format, a single-concentration screen of ∼7000 chemogenetically annotated small molecules produced a Z' factor of 0.60, an ∼18-fold signal window, and hit rates of 3.4% for inhibitors and 1.3% for activators. Concentration-response testing confirmed 95% and 57% of these, respectively. Orthogonal p-ERK HTRF assays showed high concordance with the Lumit assay, and target annotation analysis revealed enrichment for MEK, ERK, BRAF, PKC. To support plate-based HTS campaigns such as the 1536-well screen of a chemogenetic compound library, we used an alternative substrate dilution buffer that markedly improves on-deck substrate stability for at least 18 h without altering EC₅₀/IC₅₀ values or assay window, enabling reliable integration of the Lumit p-ERK assay into automated HTS workflows. Together, these data support the use of the Lumit p-ERK assay as a scalable, automation-compatible bioluminescent format for cell-based assays.