Development of EIA systems for active-form MAP kinase

Quantitative sandwich enzyme immunoassay (EIA) systems, that can distinguis h between active-form subtypes of mitogen-activated protein kinases (p44 an d p42 MAP kinase, also called ERK1 and ERK2), were developed employing subt ype-specific antibodies as a solid phase and an antibody specific for the p hosphorylated region of MAP kinases as the detector. Using these systems, w e investigated the dynamic changes in the activity of ERK1 and ERK2 in plat elet-derived growth factor (PDGF)-treated rat mesangial cells and nerve gro wth factor (NGF)-treated PC12. Both ERK1 and ERK2 were activated immediatel y after stimulation, and the activity reached a maximum at 5-10 min. The to tal activity of both subtypes correlated well with that obtained using the conventional method. Compared with the usual methods, these systems should have a higher specificity and be more convenient and suitable for experimen ts with multiple samples. Moreover, as these EIA systems can be applied not only to rat MAP kinases but also to human, mouse and rabbit MAP kinases, t hey are potentially very useful for a range of investigations. (C) 2000 Els evier Science B.V. All rights reserved.