Automation Of A Novel Cell-Based ELISA For Cell Signaling Pathway Analysis

By Wendy Goodrich and Peter Banks, BioTek Instruments, Inc., Winooski, VT and Ron Osmond and Antony Sheehan, TGR Biosciences, Adelaide, Australia

Monitoring and quantifying cell signaling pathways is critical for understanding the behavior of cell processes and many disease states. Protein kinases involved in these cellular cascades play many diverse biological roles including normal growth and development; their aberrant behavior is linked to a number of infirm states including cancer. A novel one wash step ELISA assay has been developed for detection of cell based signal transduction events. In addition to offering simplicity, speed, and flexibility, the technology is a highly sensitive technique that lends itself to automation at multiple workflow levels. To confirm optimal assay performance under full automation studies were conducted to demonstrate sound intra- and inter-assay precision, accuracy, S/B, and Z’. Subsequently, verification of a ‘one well one day’ cell-based assay protocol was developed allowing treatment and lysis of the cells directly in the ELISAOne microplate. Pharmacology assessment using this procedure was then undertaken for agonist/antagonist analysis of the PI3/AKT/mTOR signaling pathway using MCF-7 cells and multiple antagonist compounds.