A new phosphospecific cell-based ELISA for p42/p44 MAP kinase, p38 MAP kinase, c-Akt/PKB and CREB

Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed EL1SA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 MAPK, p38 MAPK, protein kinase B and the transcription factor CREB in 96 well plates. This assay has been termed PACE (Phosphospecific Antibody Cell-based ELISA) and was successfully used for both adherent and suspension cells. Various stimuli induced dose-dependent enzymatic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentrationdependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. Hence PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.