PyCSP-specific antibody responses in mouse sera were evaluated by peptide ELISA, as previously described [57] . Briefly, flatbottomed 96-well microtiter plates (Immulon 4; Dynex Technology Inc., Chantilly, VA, USA) were coated with 100 ml/well of recombinant PyCSP protein at a concentration of 1 mg/ml in carbonate-bicarbonate coating buffer pH 7.4, and incubated overnight at 4uC. Wells were blocked for 1 hr with 2% BSA in PBS containing 0.05% Tween 20 (Blocking Buffer) and washed three times with PBS containing 0.05% Tween 20 (PBS-T). Consecutive dilutions of individual sera diluted in PBS containing 0.01% Tween-20 were incubated for 2 hrs at room temperature. The plates were washed 3 times, and incubated with 100 ml/well Biotinylated anti-mouse IgG (Jackson ImmunoResearch) at a dilution of 1:20,000 for 1 hr. The plates were washed three times and incubated with Streptavidin HRP (BD Biosciences) at a dilution of 1:1000 for 1 hr. The plates were washed and developed for 10 mins with 50 ml/well TMB substrate (Sigma). Reactions were terminated by adding 50 ml of stopping buffer and the OD450 recorded using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Results are expressed as mean OD readings of triplicate wells +/-SE.