The short TgERK7 peptide, ELISA plate and SBA Clonotyping System-HRP Kit (Southern Biotech, Birmingham, USA) were used for detection of the specific anti-TgERK7 and total IgG antibodies. Briefly, ELISA plate was coated with 1.0 μg of capture antibody provided by the kit or equal short peptide diluted into the sterile PBS (pH 7.4) via incubation overnight at 4 °C. The following protocols including the antibody incubation were performed as the previous description (22 (link)). The absorbance was recorded at 450 nm at 10 min and 20 min after substrate addition using a ELISA reader (Bio-TekEL × 800, USA).
For cytokine, the levels of tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interferon-gamma (IFN-γ), interleukin 2 (IL2), IL4, IL6, IL10, IL12p70 and IL23 in sera separated at 6 weeks post the first immunization were detected by the LEGEND MAXTM Mouse ELISA kits (Biolegend, San Diego, USA). Their concentrations were calculated according to the standard curves that were drawn with the standard samples provided by the kits. The data was recorded by the reader (Bio-TekEL × 800, USA) at 450 nm within 30 min after stop buffer addition.
For cytokine, the levels of tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interferon-gamma (IFN-γ), interleukin 2 (IL2), IL4, IL6, IL10, IL12p70 and IL23 in sera separated at 6 weeks post the first immunization were detected by the LEGEND MAXTM Mouse ELISA kits (Biolegend, San Diego, USA). Their concentrations were calculated according to the standard curves that were drawn with the standard samples provided by the kits. The data was recorded by the reader (Bio-TekEL × 800, USA) at 450 nm within 30 min after stop buffer addition.