Miniaturization of a Lumit p-ERK immunoassay for cell-based high-throughput screening of a chemogenetic small-molecule library
Swiatnicki M. et al. (2026). Miniaturization of a Lumit p-ERK immunoassay for cell-based high-throughput screening of a chemogenetic small-molecule library. SLAS Technology. 40 100445. DOI: 10.1016/j.slast.2026.100445
Cell-based assays that measure endogenous signaling events are attractive for high-throughput screening, but many formats rely on engineered reporters or fluorescence detection limited by background interference. Swiatnicki and colleagues adapted the Lumit p-ERK immunoassay to higher-density plate formats suitable for automated screening. Starting from a standard 96-well protocol, they systematically optimized lysis buffer concentration and antibody volumes for both 384-well and 1536-well plates while maintaining a simple, homogeneous add-mix-read workflow. The optimization was guided by the principle of maximizing signal-to-background ratio while preserving the ability to detect basal phosphorylated ERK in MCF-7 cells.
In 384-well format, the optimized assay achieved an average Z’ factor of 0.69 and approximately 32-fold signal window between stimulated and inhibited controls—performance metrics well within the range suitable for high-throughput screening. A manually executed screen of 190 FDA-approved compounds identified six known kinase inhibitors with activity on the MAPK/ERK pathway, confirming both the specificity and sensitivity of the optimized assay. Scaling to 1536-well plates, a single-concentration screen of approximately 7000 chemogenetically annotated small molecules yielded a Z’ factor of 0.60 and 18-fold signal window, with hit rates of 3.4% for inhibitors and 1.3% for activators. Concentration-response confirmation showed 95% of the inhibitor hits were validated, and comparison with an orthogonal p-ERK HTRF assay revealed high concordance between the bioluminescent and fluorescence-based formats.
The authors also addressed a practical limitation of bioluminescent assays: substrate stability during extended automated screening runs. They tested an improved substrate dilution buffer that preserves assay window and potency measurements while maintaining stability for at least 18 hours on deck—enabling reliable integration into automated HTS workflows. Target-annotation analysis of validated hits showed enrichment for protein kinase inhibitors targeting MEK1, ERK, BRAF, and Abl-family kinases, with PKC isoforms also appearing frequently. The authors note that because PMA was used as a stimulant, a substantial fraction of hits likely act at or upstream of PKC rather than directly on MEK/ERK. These results establish the Lumit® p-ERK Immunoassay as a scalable, automation-compatible platform for high-throughput screening in drug discovery.
Keywords: p-ERK, phosphorylation, MAPK/ERK pathway, MAPK signaling, immunoassay, cell-based assay, bioluminescence, high-throughput screening, HTS, compound screening, kinase inhibitors, Lumit Immunoassay, Lumit® pERK Immunoassay, 384-well plate, 1536-well plate, MCF-7 cells.
