Tetramethylbenzidine solution

RBD or Spike protein diluted in coating buffer [1 μg ml⁻¹; 28.5 mM Na₂CO₃ and 71.4 mM NaHCO₃ (pH 9.6)] was used to coat 96-well plates for 2 hours at 37°C. Wells were washed and then blocked with 2% BSA in PBS containing 0.1% Tween 20 (PBS-T) for 2 hours at 37°C. Mouse sera (serially diluted 10-fold in PBS-T containing 1% BSA) were incubated in the wells for 1 hour at 37°C and then washed with PBS-T. Goat anti-mouse IgG-HRP (GenScript, catalog no. A00160) was added. Wells were washed again with PBS-T before adding tetramethylbenzidine solution (Thermo Fisher Scientific, catalog no. J60461). Antibody titers were defined as the reciprocal serum dilution at which the absorbance at 450 nm exceeded background by greater than 0.5 absorbance units.

Spike protein (1 μg ml⁻¹) diluted in coating buffer [28.5 mM Na₂CO₃ and 71.4 mM NaHCO₃ (pH 9.6)] was used to coat 96-well plates for 2 hours at 37°C. Wells were washed and then blocked with 2% BSA in PBS-T for 2 hours at 37°C. Mouse sera (serially diluted 10-fold in PBS-T containing 1% BSA) were incubated in the wells for 1 hour at 37°C and then washed with PBS-T. Goat anti-mouse IgG-HRP (GenScript, catalog no. A00160), goat anti-mouse IgG1-HRP (SouthernBiotech, catalog no. 1073), goat anti-mouse IgG2a-HRP (SouthernBiotech, catalog no. 1083), goat anti-mouse IgG2b-HRP (SouthernBiotech, catalog no. 1093), goat anti-mouse IgG2c-HRP (SouthernBiotech, catalog no. 1077), or goat anti-mouse IgG3-HRP (SouthernBiotech, catalog no. 1103) was added. Wells were washed again with PBS-T before the addition of tetramethylbenzidine solution (Thermo Fisher Scientific, catalog no. J60461). Antibody titers were defined as the reciprocal serum dilution at which the absorbance at 450 nm exceeded background by greater than 0.5 absorbance units.

After four-week experiment, sera were collected from BALB/c mice and stored at -80°C until analysis. 50 μl of CII (5 μg/ml) in PBS were coated onto each well of an ELISA plate overnight at 4°C. The plates were blocked using 1% BSA at 37°C for 1 h. Sera from immunized mice were loaded into the wells and incubated for 2 h at room temperature. The plates were washed, 50 μl of HRP conjugated antibodies was added to each well for 1 h at room temperature. Serum dilution was optimized. HRP activity was measured using tetramethylbenzidine solution (eBioscience). Results were analyzed by measuring the absorbance at 450 nm.
Hind paws and ankles were removed from the sacrificed mice, frozen in liquid nitrogen and lysed using RIPA buffer with protease inhibitor cocktail (Sigma chemical). The crude extract was then sonicated for 30 s. The homogenate was centrifuged at 20,000 × g for 15 min, and the resulting supernatant was collected. The levels of inflammatory factors in the supernatant were measured using specific ELISA kits according to the manufacturer's instructions.

Serum was collected 7 weeks after the first immunization for determination of the collagen-specific total IgG, IgG1, and IgG2a levels. CII (40 μg/mL) in coating buffer (0.05 M sodium carbonate anhydrous in distilled water, pH 9.6) was used to coat 96-well flat-bottom plates at 4°C overnight. Serially diluted serum samples were incubated in the wells for 1 h at room temperature. The wells were washed with washing buffer (phosphate-buffered saline containing 50 mM Tris, 0.14 M NaCl, and 0.05% Tween 20), and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was added (Bethyl Laboratories, Montgomery, TX, USA). HRP activity was assayed by adding tetramethylbenzidine solution (eBioscience) and determining the absorbance at 450 nm.

For CII-specific IgG, IgG1, and IgG2a analysis, serum was collected 70 days after the initial immunization. Flat-bottom 96-well plates were coated with a total of 40 μg/mL CII in phosphate-buffered saline (Nunc, Roskilde, Denmark) at 4 °C overnight. Serially diluted serum samples were applied and incubated at room temperature for 1 h. The plates were washed, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, or IgG2a (Bethyl Laboratories, Montgomery, TX, USA) was added. HRP activity was measured using a tetramethylbenzidine solution (eBioscience). The results were analyzed by determining the absorption at 450 nm (A₄₅₀). For analysis of TNF-α, IL-6, and IL-17 expression levels, plates were coated with anti-mouse TNF-α, IL-6, or IL-17 antibodies (all from R&D Systems) by incubation overnight at 4 °C. The plates were washed and then treated with a blocking solution for 2 h at room temperature. Cell culture supernatants and biotinylated mouse TNF-α, IL-6, and IL-17 were added; the reactions were then continued for 2 h. The plates were washed again, and ExtrAvidin-alkaline phosphate diluted 2000-fold(Sigma–Aldrich) was added. After 2 h of incubation, the samples were washed, 50 μL of p-nitrophenyl phosphate disodium salt diluted in diethanolamine buffer at 1 mg/mL was then added, and the results were analyzed by determining the A₄₀₅.