Fig.1.

Improvement in antibody affinity following chain shuffling. Inhibition ELISAs were conducted by preincubating polyclonal phage derived from two rounds of selection with a range of antigen concentrations prior to incubation on antigen-coated wells. Bound phage populations were detected with anti-M13 antibody. Signal in ELISA from these samples is expressed relative to the uninhibited control. Phage particles were derived from panning using either the original library (“Unshuffled-PAN”) or the shuffled library (“Shuffled-PAN”) or by stringent selection on biotinylated antigen (“Shuffled-Bio”). Examples are shown for the SH2 domain target VAV1 (A), the C-terminal domain of PTPN11C (B), and the ZAP70 tandem (C) and SHC1 (D) SH2 domains. Selection numbers, which are also used in clone names, are in parentheses.