3 3 5 5 tetramethylbenzidine substrate

Free OMVs or OM-NDs were administered to mice via the hock on days 0, 7, and 14 at a protein dose of 0.5 μg, and the serum from each mouse was sampled on days 0, 7, 14, and 21. Anti-P. aeruginosa titer levels were determined by a direct ELISA. OMVs dissolved in ELISA coating buffer (BioLegend) were coated overnight at 4 °C onto 96-well assay plates at 0.5 μg per well. The plates were then blocked at room temperature for 1 h with 5% skim milk (Apex Bioresearch Products) dissolved in PBS containing 0.05% Tween-20 (National Scientific) (PBST), incubated with serially diluted serum samples for 2 h, and probed with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG (BioLegend) for another 2 h. To analyze the various IgG isotypes, HRP-conjugated anti-mouse IgG₁, IgG_2b, or IgG₃ (SouthernBiotech) was used as the probe for the last step. The plates were developed with 3,3′,5,5′-tetramethylbenzidine substrate (BioLegend), and the reaction was stopped with 1 N HCl (Fisher Scientific). The absorbance was read at 450 nm with 570 nm as the reference using a TECAN Spark 20M microplate reader. All plates were washed at least 4 times with PBST between each step, and incubation steps were performed at room temperature on a rotary shaker.