Immunolon 4hbx elisa plates

ELISAs for parasite-specific IgG were performed as previously described [31 (link)]. Briefly, Immunolon 4HBX ELISA plates (Thermofisher) were coated overnight at 4°C with 5 μg soluble Toxoplasma antigen (STAg) diluted in 1X PBS. Following antigen coating, plates were washed with 1x PBS with 0.1% Triton and 0.05% Tween, then a blocking step was performed using 10% FBS for 2 hours at room temperature. After washing, serial dilutions of serum were added to plate wells overnight at 4°C. Following incubation with serum samples, plates were washed as described above and wells were incubated with goat α-mouse IgG, human ads-HRP (Southern Biotechnology) for 1 hour at room temperature. Finally, ABTS peroxidase substrate solution (KBL) was added. Immediately after a color change occurred plates were read on an Epoch BioTek plate reader using Gen5 2.00 software.

Samples for ELISAs were obtained by harvesting mouse brains and processing them to form a single cell suspension. Cells were then plated in 96-well plates and incubated at 37 °C either overnight or for 5 h (indicated in figure legends). Supernatants were then collected and stored at −20 °C until use. ELISAs for IL-1α (BioLegend 433401) and IL-1β (BioLegend 432601), as well as for IFN-γ, were performed according to the manufacturer’s instructions. Briefly, Immunolon 4HBX ELISA plates (Thermo Fisher Scientific) were coated with capture antibody at 4 °C overnight. Plates were then washed and blocked with buffer containing BSA at room temperature for 1 hour. After washing, standards and samples were added and incubated at room temperature for 2 h. After washing, biotinylated detection antibody was added and incubated for 1 h at room temperature. Plates were washed and incubated with avidin-HRP for 30 min at room temperature. Finally, plates were washed and incubated with ABTS peroxide substrate solution (SouthernBiotech) for 15 min or until color change occurred. Immediately after the color change, plates were read on an Epoch Biotek plate reader using Gen5 2.00 software.