Enspire 2300 elisa reader

The serums were treated with magnetic beads coupled with DTT to remove antibodies against DTT, and then the antibody titer and absorbance (1:200 dilutions) after each treatment were detected by ELISA. Secondary antibodies used were goat anti-mouse IgG-HRP, or goat anti-mouse IgG1-HRP, or IgG2b-HRP, or IgG2c-HRP, or IgG3-HRP, or IgM-HRP (1:5000 dilutions, Shanghai Immune Biotech Co. Ltd., Shanghai, China), and 3, 3′, 5, 5′-tetramethylbenzidine (TMB, TIANGEN, Beijing, China) as the substrate agent. The absorbance at 450 nm was measured by EnSpire 2300 ELISA reader (PerkinElmer, Waltham, MA, USA).
The amount of IFN-γ secretion in the supernatants diluted 2-fold from ex vivo stimulation above were measured by sandwich ELISA using the mouse IFN-γ DuoSet ELISA kits (R&D Systems) following the manufacturer’s instruction. All standards and samples were assayed in duplicate and data were analyzed in Microplate Reader with a 6-parameter fit for the standard curve.
To assess inflammatory reactions, IL-6 and IL-8 concentrations of serum from mice treated with DTT-COS1, DTT-COS2, DTT-COS12, or PBS were measured by Mouse IL-6 ELISA Kit (BOS-20268) and Mouse IL-6 ELISA Kit (BOS-46967) following the manufacturer’s instruction. The absorbance at 450 nm was measured by EnSpire 2300 ELISA reader (PerkinElmer, Waltham, MA, USA).

To assess liver injury, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations of serum from mice treated with DTT-COS1, DTT-COS2, DTT-COS12, or PBS (in the presence of aluminum hydroxide Gel adjuvant and CpG ODN 1826, as mentioned in 2.4.) were detected using the kit (Jiancheng Biologic Project Co., Nanjing, China) following the manufacturer’s instructions. The absorbance at 510 nm was measured by EnSpire 2300 ELISA reader (PerkinElmer, Waltham, MA, USA).

To detect antibodies against neoAg, ELISA plates were incubated overnight at 4°C with 100 ng CTB-neoAg, or DTT, or CTB-wtAg in 100 μL sodium carbonate buffer, pH 9.6. The non-specific binding sites were blocked with 100 μL 3% skim milk in PBS + 0.05% Tween 20 at room temperature for 1 h. Subsequently, the mouse sera with indicated dilution were added to the wells and incubated for 1 h at room temperature. The bound antibodies were detected using goat anti-mouse IgG-HRP, or goat anti-mouse IgG1-HRP, or IgG2a-HRP, or IgG2b-HRP, or IgG3-HRP, or IgM-HRP (1:5,000 dilutions, Shanghai Immune Biotech Co. Ltd., Shanghai, China) using 3,3′,5,5′-tetramethylbenzidine (TMB, TIANGEN, Shanghai, China) as substrate. The absorbance at 450 nm was measured by EnSpire 2300 ELISA reader (PerkinElmer, Waltham, MA, USA). The antibody titers are defined as the reciprocals of the highest dilution yielding an optical density of 0.2 or greater than that of pre-immune mouse sera.