A 1536-COLORIMETRIC SPAP REPORTER ASSAY - COMPARISON WITH 96-WELL AND384-WELL FORMATS

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