10 6 ml the cells are incubated with the test inhibitor compounds for 1 hour at 37 u00b0 c following this incubation a 20 microliter quantity of the fully complexed bead suspension at a bead density of 2 5 u00d7 10 7 ml in supplemented rpmi is added to each test well as a result 5 u00d7 10 5 beads well are used and the incubation is continued for 18 hours at 37 u00b0 c the supernatants from each well are transferred to 96 well v bottom plates in order to pellet the cells and beads the supernatants are then assayed for interleukin 2 with the human il 2 kit r amp d d2050 according to instructions contained therein the assay does not specifically discriminate between inhibition of t cell activation caused by inhibition of ick zap 70 and itk ptk enzymes or any combination thereof but serves as a useful screen of promising compounds data are analyzed by polynomial regression and analzyed using a macro program an ic 50 value of less than about 5 u03bcm is preferred screen for immunosuppressive compounds that inhibit the kinase activity of ick enzyme in the following assay the potency of a test compound is determined as an ic 50 value that is the concentration of compound needed under assay conditions to inhibit 50 of ick phosphorylation activity in the present assay the ick substrate is u201c pgt u201d poly glu tyr as sodium salt the following reagents are used in the assay g nunc maxisorp plates van waters amp rogers catalog no 62409 004 h ick gst enzyme a fusion protein of ick glutathione u2014 s transferase expressed from a baculovirus vector system and purified on a glutathione affinity column k phosphorylation buffer 50 mm hepes ph.