AMA1 protein products
were identified using 4G2 monoclonal antibody or rabbit polyclonal antiserum raised against the Reduced Alkylated AMA1 protein. MSP1 protein products were identified using MSP1-specific polyclonal antibody R94256. T cell responses were assessed by ELIspot using splenocytes harvested at 2 or 6 weeks post-immunization and A20 cell targets transfected with plasmid DNA using the AMAXA nucleofector system (AMAXA Inc., Germany). Briefly, multiscreen MAHAS 4510 plates (Millipore, Bedford, MA) were coated AUY-922 with 100 μl/well of sterile PBS (pH 7.4) containing 10 μg/ml of anti-murine IFN-γ (clone R4-6A2, Pharmingen, San Diego, CA) and incubated overnight at room temperature. Plates were washed twice with 200 μl/well
RPMI medium and blocked with 200 μl/well of cRPMI medium (RPMI-1640 with 10% FCS, 25 mM Hepes, l-glutamine, and Penicillin-Streptomycin) in 5% CO2 at 37 °C for at least 3 h. After blocking, the plates were washed once more with cRPMI before the addition of target and effector cells. To obtain target cells, A20.2J (ATCC clone HB-98) target cells were transfected using the AMAXA Nucleofector Kit V kit with commercially produced (PureSyn, Malvern, PA) plasmid DNA encoding VE-821 purchase PfAMA1 (VR2577), PfMSP142 (VR2574) or plasmid DNA without insert (VR1020), according to manufacturer’s protocol 18 h prior to assay, washed once with cRPMI, irradiated in a 137Cs gamma irradiator (16,666 rads), washed 3 times with cRPMI, and diluted to 1.0 × 106 cells/ml (A20.2J) in cRPMI. much To obtain effectors, single cell suspensions were prepared from harvested splenocytes, washed 3 times, counted, and diluted to 10 × 106 cells/ml;
a pooled splenocyte preparation was made for each group (6 mice/group). Effector and target cell preparations were added to the IFN-γ coated wells in quadruplicate at 100 μl/well, and incubated in 5% CO2 at 37 °C for 36 h. Plates were flicked to remove the cells and washed 6 times with PBS-T (PBS 0.05% Tween-20). Then 100 μl/well of biotinylated anti-IFN-γ (clone XMG1.2, Pharmingen, San Diego, CA) at 2 μg/ml in PBS-T was added to the plates which were incubated overnight at 4 °C. Plates were washed 3 times with PBS-T and 100 μl/well peroxidase conjugated streptavidin (Kirkegaard & Perry, Gaithersburg, MD) was added at 1:800 dilution in PBS-T. After 1 h incubation at room temperature, plates were washed 3 times with PBS-T followed by 3 times with PBS alone, and developed with DAB reagent (Kirkegaard & Perry) according to manufacturer’s instructions. After 15 min, the plates were rinsed extensively with dH2O to stop the enzymatic reaction, dried and stored in the dark. Spots were counted with a KS ELIspot reader (Carl Zeiss, Vision, Germany).