Vaccination with recombinant adenovirus expressing multi-stage antigens of Toxoplasma gondii by the mucosal route induces higher systemic cellular and local mucosal immune responses than with other vaccination routes

Wang, Ting; Yin, Huiquan; Li, Yan; Zhao, Lingxiao; Sun, Xiahui; Cong, Hua

Serum samples were collected by retro-orbital bleeding on days 0, 14, 35, and 49. Standard ELISAs were used to determine the levels of T. gondii-specific antibodies, IgG, IgG1, IgG2a, and IgA, in the serum samples from the inoculated mice. This was done with some changes to the method previously described [3, 5] . Briefly, a flat-bottom 96-well plate was pre-coated with the UMAS peptide pool at a concentration of 10 lg/mL in a 50 mM carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. The mouse sera were diluted in PBS (1:100) and incubated at 37°C for 1 h. After washing the plates, bound antibodies were then reacted with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgA (Sigma-Aldrich, USA) at 37°C for 1 h. Peroxidase activity was revealed by 3, 3 0 , 5, 5 0 -tetramethylbenzidine (TMB, 10 mg/mL) and stopped by adding 50 lL of 2 M H 2 SO 4 . The optical density (OD) at 450 nm was measured by a Thermo Scientific Multiskan FC Microplate Photometer (Thermo Scientific, USA).