Evaluating T. gondii Antibody Titers and Cytokine Profiles

To investigate the titers of T. gondii-specific serum antibody in the sera, ELISAs were conducted as described previously (44 (link)). Briefly, each well (96-well plates, Costar, Cambridge, MA, USA) was coated with 1 μg of STAg (dissolved in 100 μl of carbonate buffer pH 9.6) overnight at 4°C. After 5-min rinsing in TBST, each well was blocked with TBST containing 5% BSA (Sangon Biotech, Shanghai, China) at 37°C for 1 h. Sera from mice were diluted (1:100) in TBST containing 5% BSA and added into each well after being rinsed in TBST for 5 min. Incubated at 37°C for 1 h, each well of the 96-well plates was rinsed 5 min in TBST and incubated with HRP-conjugated anti-mouse IgG, IgG1, or IgG2a (1:8,000, eBioscience, San Diego, USA) at 37°C for 1 h. Finally, tetramethylbenzidine (TMB; Tiangen, Beijing, China) as substrate was used to evaluate the immunoreaction, and the reaction in each well was stopped by 100 μl of 2 M newly prepared H₂SO₄. The absorbance was determined at 450 nm by a microplate reader (Thermo Scientific, Waltham, MA, USA).
To determine cytokine secretions in animals’ sera, commercially available ELISA kits (Yifeixue Biotech, Nanjing, China) based on double antibody sandwich method were used to evaluate the concentrations of interferon-gamma (IFN-γ), interleukin (IL) 4 (IL-4), IL-10, and IL-17 referenced to known amounts of mouse recombinant cytokines.