To further examine the ‘‘specificity’’ of the affinity-selected phages, representative phages from each collection were hybrid- ized with each of the 3 polysera using dot blot. In each case, the selected phage hybridized best with the sera used for the affinity Table 1. Mimotope sequences of affinity-selected phage. HER-2ECDa mimotopes HER-2FL mimotopes HER-2TUBO mimotopes Mimotope Phage sequence Mimotope Phage sequence Mimotope Phage sequence SRCDLDEKGC TUBO.7 SPPAFSHPMQ YMDPHTQREA FL.8 PRCKHNQKKC TUBO.8 VNTQGPNSIA NWSIAGD SRGDTEP TSDSWQR PKTEVPQ DDFSPPR EWYTPQG AGACKRMREF ATPKRTHDHD TSNNRVEARA RTTPSGGGFK DGWYVAQ GGRWGES WYTTPGS SGWYTPV AKDEQPM GWYVSSP PSEGQSE LQEFPGDQLV LSTRLWIPAW KTTTWPSTPT MWASVNKMA PPPLLAGDPK AVNQCTTVLA GTAGGEITEHE TUBO.1 TYSGRAACGV TUBO.2 SASMPFPANE TUBO.3 SKPKYEKPSQ TUBO.4 EVTTEHVEAN TUBO.5 RSHERHPPSP TUBO.6 DDELHSGTSY Epitope Mapping with Mimotopes Figure 1. Antigen structure influences the composition of the polyclonal response. A. ELISA plate wells were coated with recombinant rat ECD and reacted with serial dilutions of mouse sera. B. Recombinant extracellular domain (ECD) of rat HER-2 was treated (reduced protein) or not (native protein) with b-mercaptoethanol and run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1:1000. C. 106 Tubo cells over-expressing rat HER-2 were reacted with serial dilutions of mouse sera, probed with anti-mouse antibody conjugated with PE and fluorescence intensity was measured by flow cytometry. Each point is reflective of at least 10 000 events. D. Recombinant extracellular domain of rat HER-2 or human HER-2 were run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1:1000 or anti-HER-2 antibody. E. 500 TUBO cells were plated into 96 well plate in DMEM medium supplemented with 5% FBS and incubated 1–2 weeks in the presence of IgGs purified from mouse sera at a final concentration of 25 mg/ml.. The cell proliferation was measured according to manufacturer instruction by CellTiter 96H Aqueous Non-Radioactive Cell Proliferation Assay. The error bars reflect the mean+/2sem for 4 samples. These data represent the results of pooled serum from 5 vaccinated mice per group. Each experiment was replicated at least 3 times and representative results are shown. doi:10.1371/journal.pone.0005309.g001 purification (Figure 2). Even though we did not isolate mimotopes using HER-2FL serum that matched the sequence of the mimotopes selected with HER-2ECD serum, we did find 2 phage isolated with HER-2FL that could be recognized by both sera (FL.6 and FL.23) (Figure 2A). Notably, phage FL.23 was bound by all 3 sera. In the case of the phage isolated with HER-2ECD serum, although we observed low-level cross-reactivity with HER-2FL serum, we observed no cross-reactivity with HER-2TUBO serum (Figure 2B). With regard to the phage selected by HER-2TUBO serum, comparable, low-level cross-reactivity was seen with both HER-2FL and HER-2ECD (Figure 2C). Given the limited cross-reactivity between the affinity-selected phages, it appeared as though the selected phage may reflect a molecular ‘‘signature’’ of the polyclonal specificity. One way to describe such a ‘‘signature’’ would be the analysis of the specific amino acid residues within each collection of phages. Each collection of phages was dominated by a distinct set of amino acid residues (Table 2) resulting in a distinct hierarchy of amino acid frequency with the various collections. Furthermore, when we examined the frequency of specific amino acid pairs, which reflects the minimal homology elements that are employed by the computer algorithm for epitope prediction [9,11], we found that the majority of generalized pairs were unique to each collection (Table 3, bold letters) and only a few pairs were found in more than 1 mimotope collection (Table 3, italicized). These results demonstrate that the phage selected through our affinity- purification method represent a ‘‘signature’’ of the respective polyserum used for purification. Affinity-selected phage can be used to predict epitopes targets of the polyserum Of interest, we also sought to determine whether the affinity- selected mimotopes could be used to elucidate the epitopes recognized by the polyserum. To attribute the mimotope sequences to specific epitopes on the protein, we used our novel computer algorithm [9] which categorizes mimotopes based on minimal homologies (i.e. neighbouring amino acids) into clusters defined by the position of these minimal homologies on the folded protein. These clusters represent hypothetical epitopes. As a result of cluster analysis performed using stringent conditions where we only included amino acid pairs determined to be significant based on their actual occurrence compared to the estimated random occurrence [11], a numbers of distinct clusters were found on the surface of the protein (Figure 3, Table 4). Six epitopes were predicted for the HER-2ECD polyserum (ECD#1–ECD#6; Figure 3A; Table 4), six epitopes were predicted for the HER-2FL serum (FL#1–FL#6; Figure 3B; Table 4) and a single epitope was predicted for HER-2TUBO serum (TUBO#1). To determine whether the epitopes predicted by our affinity- selected mimotopes are consistent with regions predicted to be immunogenic, we employed the DiscoTope computer algorithm ECD.1 ECD.2 ECD.3 ECD.4 ECD.5 ECD.6 ECD.7 ECD.8 ECD.9 ECD.10 ECD.11 ECD.12 ECD.13 ECD.14 ECD.15 ECD.16 ECD.17 ECD.18 ECD.19 ECD.20 ECD.21 ECD.22 ECD.23 ECD.24 ECD.26 ECD.27 ECD.28 ECD.29 ECD.30 ECD.31 FL.1 FL.2 FL.3 FL.4 FL.5 FL.6 FL.7 FL.9 FL.10 FL.11 FL.12 FL.13 KAEERFHEVR WNIFQSLSSP RVAATAAREE TPEPLEEAAS DHVFKRPQPS ETCVEKNEAD WRRDGC EPVRDNCAPS VERDIATRPW ERPEIEDVCQ QKTLNTSNAN VKTLNTSNAN RDTTMWEVNA FL.14 WGHCSQGMIE FL.15 PPFDVFHNPM FL.16 DTHAGMHSPT FL.17 RMPHHDPQLM FL.18 TQNLMQMQHA FL.19 TCQAGRESMHNP FL.21 FYSSMFWAVGEQ WETMHNPGTP FL.22 DALNMHEGRP FL.23 SKLETTMHSP FL.24 NTAHSDMHSP FL.26 MAQMHEPVRS FL.27 NTAHSDMHSP FL.28 LPQHNMMHDP PSNYSAMHAP RCETHFNMHEPY ECD.25 LGGMDSMHSP FL.25 aPolyserum used for mimotope isolation. doi:10.1371/journal.pone.0005309.t001 SPFMLMHGEH FL.20 GTAGGEITEH PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5309