Mouse Anti-TvAP33 Antibody Detection

The collection of mouse blood samples in each group (n = 5) was conducted at 0, 14, 28, and 42 days, and the obtained serum samples were stored at −20°C in order to further evaluate antibodies and measure cytokines. Indirect ELISA was performed to detect IgG isotypes and specific anti-TvAP33 antibodies as described in a previous study with a few modifications (Zhang et al., 2018c (link)). In brief, microtiter plates (Costar, New York, NY, United States) were coated with recombinant TvAP33 in carbonate buffer (2.5 μg/ml, 100 μl/well) with a PH value of 9.6 overnight at 4°C and blocked with 4% BSA for 2 h at 37°C. A mouse serum dilution (ratio = 1/10, PBS was used as the diluent) was added to the wells, and the plates were incubated at 37°C for 2 h. After the plates were washed three times with PBST, the plates were treated with the HRP-conjugated secondary antibodies goat anti-mouse IgG2a, IgG1, and IgG (SouthernBiotech, Birmingham, AL, United States). Finally, 100 μl of 3,3,5,5-tetramethylbenzidine was added into each well, and then 100 μl (2 M) sulfuric acid was added to terminate the reaction. The light absorption at 450 nm was measured by an automatic ELISA reader (MULTISKAN FC, Thermo Fisher Scientific, Waltham, MA, United States) and all tests were completed in triplicate.