Assessment of humoral immunity

Serum samples were obtained from five mice per group 2 days before each injection and 15 days after the last immunization. The specimen was collected and stored at a temperature of − 70 °C. The identification of serum G1 (IgG1) and secretory IgA (sIgA) from fecal pellets, with specificity to Omp10, was accomplished using an indirect ELISA, according to a pre-established protocol. The wells of a polycarbonate plate were coated with 100 μL of a 5 μg/mL solution of pure Omp10 in carbonate buffer (pH 9.6). After washing with PBS containing 0.05% (w/v) Tween 20, the plates were incubated with 5% skim milk in PBS containing 0.5% Tween 20 for an additional overnight at 4 °C to avoid non-specific binding. After removing the blocking fluid, the plates were incubated with 100 µL of blood samples diluted 1:100 in preventing buffer for 2 h at room temperature while gently rocking. The wells were then incubated at room temperature for an additional hour with a dilution of 1:2000 Goat Anti-Mouse IgG (Bio-Rad Cat No: 170-6516) tagged with HRP. Three further washes with PBS-Tween were performed between incubations. After a final wash, specific reactivity was determined by adding 50 µL/well of the enzyme–substrate TMB and incubating the plates at 37 °C for 30 min. The reaction in each well was stopped by adding 2 M H₂SO₄. The optimal density was calculated at 492 nm after 10 min (OD₄₉₂). Each experiment was performed three times.

Mucosal IgA (sIgA) levels were determined by analyzing fecal pellets. Using a balance, we measured and homogenized the fecal pellet samples to a final concentration of 100 mg per 0.5 mL of PBS 1 (pH 7.2) containing 1% BSA and collected them 2 weeks following the last vaccine. The samples were then incubated for 16 h at 4 °C, centrifuged for 5 min at 15,000 RPM at 4 °C, and the supernatants were utilized to detect sIgA.