A 1536 colorimetric SPAP reporter assay: Comparison with 96- and 384-well formats

We report the successful miniaturization of a functional cell-based reporter gene assay. Utilizing interleukin-1beta (IL-1 beta)-induced secreted placental alkaline phosphatase (SPAP)-catalyzed colorimetric readout, we reduced the assay volume to 10 mu l using a Greiner 1536-well microplate. Our experiences of assay development, liquid handling (using a Hydra(R) 96; Robbins Scientific, Sunnyvale, CA), and detection (using the SpectraImage and SpectraFluor-Plus plate readers, Tecan Austria GmbH, Grodig, Austria) in 1536 wells are discussed. The effect of a set of 1,280 compounds in this SPAP reporter assay were compared between 96-, 384-, and 1536-well formats and were shown to be very similar. We conclude that cell-based reporter gene assays using SPAP-catalyzed color readouts are sensitive and highly reproducible in 1536-well plates and should be considered as a cost-effective alternative to luciferase reporters for miniaturized assay formats. Finally, we review the prospects for the implementation of routine HTS in 1536-well plates based around the instrumentation investigated.