The synthetic peptide sequences were aa 232–246 (GTVQRWEKKVGEKLS), aa 236–250 (RWEKKVGEKLSEGDL), aa 240–254 (KVGEKLSEGDLLAEI), aa 244–258 (KLSEGDLLAEIETDK), aa 248–262 (GDLLAEIETDKATIG), aa 252–266 (AEIETDKATIGFEVQ), aa 256–270 (TDKATIGFEVQEEGY) and aa Selleckchem INCB018424 260–274 (TIGFEVQEEGYLAKI), all purchased from Genenet (Fukuoka, Japan). AMA was determined by ELISA using the triple-expression hybrid clone, pML-MIT-3 (pML-MIT-3-ELISA)

[10,16,17]. Briefly, recombinant proteins containing the AMA-reactive immunodominant epitopes localized to the three distinct lipoyl domains of human pyruvate dehydrogenase complex (PDC)-E2 [18], bovine branched chain 2-oxo acid dehydrogenase complex (BCOADC)-E2 [19] and rat 2-oxoglutarate dehydrogenase complex (OGDC)-E2 [10] were cloned and co-expressed in the plasmid vector, selleckchem pGEX-4T-1 (Pharmacia, Alameda, CA, USA) and the product used as antigen. Serological AMA was determined using serum samples at a 1:250 dilution and the bound antibodies were detected by peroxidase-conjugated goat anti-mouse immunoglobulin (diluted 1:50 and 100 ul/well; Dako, Glostrup, Denmark). The optical density (OD) was determined using a microplate

reader at 450 nm. Splenic mononuclear cells were obtained from mice before and at 6, 12, 18 and 24 weeks post-immunization and were treated with either NK1·1 antibody (n = 8 each time) or with control immunoglobulin (n = 8 each time) or negative control (n = 3 each time). A total of 1 × 106 cells were dispensed into each well of a 24-well plate and cultured with murine PDC-E2

synthetic peptides, as mentioned below. After 3 days of culture, viable splenocytes were harvested and ELISPOT assays were performed [RSD ELISPOT set, mouse interferon (IFN)-γ ELISPOT set, Minneapolis, MN, USA]. Briefly, 96-well nitrocellulose plates were coated with an optimized capture monoclonal antibody (mouse anti-IFN-γ) in phosphate-buffered saline (PBS) and incubated overnight at 4°C. Unbound antibody was removed by washing with PBS containing 0·05% Tween (PBS-Tween). Viable Rucaparib molecular weight cells were added at 3 × 105 cells/well in 100 µl RPMI-1640 in triplicate. The plates were incubated at 37°C, 5% CO2 for 24 h; the plates were then washed, labelled with biotin-labelled anti-IFN-γ and developed by incubation with streptavidin–alkaline phosphatase, followed by incubation with a final substrate solution (BD™ AEC substrate reagent set, San Diego, CA, USA). The reaction was stopped by rinsing the contents with distilled water, and the number of spots was counted by using a KS ELISPOT Reader (Zeiss, Thornwood, NY, USA). Known positive and negative samples were included throughout.