Tetramethylbenzidine tmb

Sera, intestinal mucus, and fecal samples were prepared on days 0, 7, 14, 21, 28, 35, and 42 after immunization and used for detecting specific IgA or IgG antibodies by ELISA. Briefly, polystyrene microtiter plates were, respectively, coated overnight at 4°C with 10 μg of C. perfringens α, β2, ϵ, and β1 toxoid proteins that were expressed by E. coli and purified in our lab. Next, the plates were washed thrice with PBS containing 1% Tween-20, and then saturated with PBS containing 5% skimmed milk at 37°C for 2 h. The sera, intestinal mucus, and fecal extraction were used as the primary antibody, and horseradish peroxidase-conjugated goat anti-mouse IgA or IgG (Sigma, USA) was used as a secondary antibody, and followed by color development using tetramethylbenzidine (TMB) (TIANGEN, China) as the substrate, and then absorbance was measured at 450 nm. Moreover, the levels of cytokines IL-4, IL-12, IL-10, IL-17, IL-2, and IFN-γ in the sera samples were detected by ELISA Kits according to the manufacturer’s instructions (Biosource International, USA).

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.

The standard checkerboard titration procedure was used to assess the optimal concentrations of the rEg-TSP11 antigen and serum. Carbonate buffer (0.1 M, pH 9.6) was used to dilute the purified rEg-TSP11 protein to 5 μg/mL, which was used as the antigen in the ELISA test. The diluted antigen solution was used to coat ELISA plates at 4°C overnight. The next day, the plate was washed with PBS-Tween-20 (PBST) and then incubated with 5% skim milk at 37°C for 2 h. After thorough washing, 100 μL of serum samples (2-fold dilutions, 1:80) in PBST was added to each well and incubated for 1.5 h at 37°C. After washing, HRP-labeled rabbit anti-dog IgG (1:3,000 dilution; Solarbio, Beijing, China) was added to the plates and incubated for 1.5 h at 37°C. After washing, the substrate, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (Tiangen, Beijing, China) was added to the wells and incubated for 1.5 h at 37°C. Finally, 1 M H₂SO₄ (100 μL) was added to stop the development of color, and the optical density was measured at 450 nm (OD 450).