Fig.2.

Primary ELISA screen of chain-shuffled antibodies selected using each of the eight different SH2 domains. Affinity selections against the different SH2 domains with the eight chain-shuffled libraries took place under different conditions, and the optimal selections were determined based on polyclonal ELISA results (data not shown). Populations were subcloned into an optimized expression vector, and 96 clones were picked and screened by ELISA. Binding of the scFvs to the immobilized SH2 domain was quantified using europium-labeled anti-FLAG secondary antibody. A graph is shown for each of the 12 selections (A–L), plotting the time-resolved fluorescence signal in intensity units (y axis) for each scFv (x axis). The graph titles indicate the selection conditions employed (e.g., PAN1–PAN2 indicates two rounds of selections were performed with antigen immobilized on polystyrene wells, Bio100–Bio10 indicates that the first selection round was performed with 100 nM biotinylated antigen and the second round was performed with 10 nM biotinylated antigen). The selection numbers, used to name the antibodies, are shown in parentheses.