3 3 5 5 tetramethylbenzidine substrate solution

Vaccine-specific IgG and IgA levels were measured from serum and specific secretory IgA levels were measured from the feces of immunized and control mice from each model of vaccination by using an antibody enzyme-linked immunosorbent assay (ELISA). ELISA plates (Corning-Costar) coated at 4°C overnight with 100 µl of 0.5-µg/ml vaccine supernatant were washed three times with PBS–0.05% Tween 20 (PBS/T) prior to blocking with 100 µl PBS–2% bovine serum albumin (BSA)–0.05% Tween 20 (PBS/BSA/T) for 1 h at room temperature. Dilutions of mouse serum in PBS–0.5% BSA were added to the plates (100 µl) and incubated at 37°C for 2 h prior to washing three times with PBS/T. To detect total IgG or IgA, 100 µl of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or IgA (1:1,000; Santa Cruz Biotechnology) was added, and the plates were incubated for 1 h at 37°C. After three washes in PBS/T, the reaction mixture was developed with 100 µl of 3,3′,5,5′-tetramethylbenzidine substrate solution (BD Biosciences) for 20 min and stopped with 50 µl of 3 M H₂SO₄. The OD₄₅₀ was measured.

Cell enzyme-linked immunosorbent assays (ELISAs) were performed as described previously, with minor modifications.^{31 (link)} Briefly, 1 × 10⁴ HUVECs plated in wells of a 96-well plate were cultured in EGM containing 20 ng ml⁻¹ of hTNFα for 24 h at 37 °C. Following fixation with 4% PFA, cells were incubated with various concentrations (0–50 μg ml⁻¹) of VCAM-1-D6-Fc-HRP or Fc-HRP for 2 h at 37 °C. For competition cell ELISA, HUVECs cultured in EGM containing 20 ng ml⁻¹ of hTNFα were incubated with 3 μg ml⁻¹ VCAM-1-D6-Fc-HRP in the absence or presence of increasing concentrations of anti-VCAM-1-D6 IgG for 2 h at 37 °C. After three washes with ice-cold PBS, the cells were incubated with 3,3′,5,5′-tetramethylbenzidine substrate solution (BD Biosciences). Optical density was measured at 450 nm using a VICTOR X4 plate reader.