Table 3. Occurrence of statistically significant amino acid pairs within mimotope collections. HER-2ECDa mimotopes Generalized Amino Acid Pairsb HER-2FL mimotopes HER-2TUBO mimotopes Occurrence within mimotope collectionc Generalized Amino Acid Pairs Occurrence within mimotope collection Generalized Amino Acid Pairs Occurrence within mimotope collection MHd JO OP BJ HO JP BP HJ OM AX XM XA JA GM HX ZY AH 11 8 8 7 7 6 5 5 5 4 4 4 4 2 2 2 2 OO OP GJ OJ AG ZY GG PB GZ JZ PP JH XX PM 10 9 7 7 5 5 5 5 4 4 4 3 3 2 Epitope Mapping with Mimotopes OY PA AX OH BP HP PP MX PM MP 2 2 2 2 2 2 2 1 1 1 aPolyserum used for mimotope isolation. bSingle letters amino acid codes (See Methods). cNumber of mimotopes carrying this generalized pair within the specific collection. dUnique pairs are presented in bold, pairs shared by two datasets are presented in italics. doi:10.1371/journal.pone.0005309.t003 the rat neu oncogene [37]. TUBO cells grow progressively in normal BALB/c mice and give rise to tumors which are histologically similar to those in BALB NeuT mice. 106 TUBO cells were injected subcutaneously into Balb/c mice and the recipients were monitored for tumor growth. For the experiments described in our manuscript, serum was obtained from peripheral blood isolated one month after tumor cell inoculation. Proliferation assay 500 TUBO cells were plated into a 96 well plate in DMEM medium supplemented with 5% FBS and incubated for 1–2 weeks in the presence of serum IgGs [purified on Protein G-Sepharose (GE Healthcare Bio-Sciences AB, Uppsala, Sweden)] diluted at final concentration of 25 mg/ml. Cell proliferation was measured using the CellTiter 96H Aqueous Non-Radioactive Cell Prolifer- ation Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Production of recombinant HER-2 Recombinant HER-2 was used for Western blot and ELISA. To provide a source of recombinant HER-2, the extracellular domains (without the transmembrane domain) of rat and human HER-2 were subcloned into pcDNA3.1-002 [35] to yield prHER- 2ECD and phHER-2ECD. The plasmids were subsequently transfected into 293T cells using Lipofectamine2000 (InVitrogen). Supernatants were collected and the samples were concentrated using a Vivaspin 50000 column (Sartorius AG, Goettingen, Germany). We also subcloned the cDNAs into pCMVhygro which encodes the hygromycin resistance gene downstream of an IRES to yield pCMVhygro-rHER-2ECD and pCMVhygro-hHER- 2ECD. These plasmids were transfected into CHO cells and stable transfectants were selected using 250 mg/ml of hygromycin (Roche Diagnostics, Mannheim, Germany) and then cloned by limited dilution. CHO cells expressing high levels of HER-2 ECD were identified by Western blot of culture supernatants. Similar to the 293T-derived HER-2 ECD, supernatants from CHO cells expressing high-levels of HER-2 ECD were concentrated by centrifugation on Vivaspin 50000 columns (Sartorius AG, Goettingen, Germany). Western blotting Recombinant HER-2 proteins from the 293T cultures were mixed with gel-loading buffer. For some experiments, the proteins were also treated with b-mercaptoethanol. The proteins were electrophoresed through 10% SDS-PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1:1000. Bound antibody was detected using goat anti-mouse IgG conjugated with HRP (Jackson Immunoresearch Laboratories Inc. West Grove, Pennsylvania, USA) and the ECL chemiluminescent reagent (Amersham Biosciences, NJ, USA). As positive controls for HER-2 detection, we employed a monoclonal antibody against rat HER-2 (BD Biosceinces, catalog# 610161) and a polyclonal serum against human HER-2 (R&D Systems, catalog #AF1129) Measurement of HER-2 specific antibodies by ELISA 96 well plates (Nunc, New York, USA) were coated with rat HER-2 produced from the CHO cultures. Wells were blocked with 5% non-fat milk in Tris-buffered saline (TBS, pH 7.4), and reacted with serial dilutions (in TBS/0.5% non-fat milk) of mouse sera. The signal was developed with substrate Sigmafast OPD (Sigma Aldrich, St. Louis, USA) after incubation with goat anti- mouse IgG conjugated with HRP. PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5309