Epitope Mapping with Mimotopes Figure 2. Limited cross-reactivity between phage selected with different polysera. Selected phage were immobilized onto nitrocellulose membranes and reacted with three different sera overnight at 4uC (HER-2FL – serum prepared from mice immunized with full-length HER-2; HER-2ECD – serum prepared from mice immunized with soluble protein; HER-2TUBO – serum from tumor bearing mice). Bound antibodies were detected with goat anti-mouse IgG HRP conjugated antibody and the signals were developed by ECL. A. phages selected with HER-2FLserum, B. phages selected with HER-2ECD-serum, C. phages selected with HER-2TUBO -serum). doi:10.1371/journal.pone.0005309.g002 to predict B-cell epitopes based on protein structure [26] (Figure 4A and Table 5). Regions predicted to be highly immunogenic are shown in red and regions predicted to be moderately immunogenic are shown in yellow. Strikingly, most of the epitopes predicted by our method mapped to regions predicted to be immunogenic based on independent criteria (Figure 4A) which offers additional confidence in the epitopes predicted by our method. The C-terminus of the HER-2 ECD was predicted to be strongly immunogenic by DiscoTope, yet none of the polyserum mapped to this site. Interestingly, this site exhibits 100% homology between mouse and rat proteins (Figure 4B). In general, the cluster analysis indicates that antibody responses are directed against epitopes on rat HER-2 where there are differences between rat and mouse sequences. Clusters ECD#1 and FL#1 are both located in a similar region of domain 1 close to N103 (this residue is a putative glycosylation site in mouse HER-2 but not in rat PLoS ONE | www.plosone.org 5 April 2009 | Volume 4 | Issue 4 | e5309