Miniaturization and automation of an ubiquitin ligase cascade enzyme-linked immunosorbent assay in 1,536-well format.

Enzyme-linked immunosorbent assays (ELISAs) are a long established and widely used assay format for drug discovery and diagnostics. They offer many advantages over homogeneous assay formats, including high sensitivity and separation (wash) steps that remove detection-interfering compounds. Many high-throughput screening assays are now performed in miniaturized formats (1,536- and 3,456-well plates) for higher throughput and lower reagent consumption. With miniaturization, separation steps in assays such as ELISA can become difficult to implement. Here we report on the implementation of the Kalypsys, Inc. (San Diego, CA) 1,536-well plate washer to enable the successful miniaturization and full automation of an ELISA that monitors ubiquitin ligase activity. The 1,536-well plate ELISA was robust and used for the high-throughput screening of a large screening collection (>1 million compounds).