Antigen-specific antibody responses by ELISA For determination of antibody responses, serum samples collected from experimental groups of mice before and after infection were analyzed for the presence of LAg-specific immunoglobulin by ELISA. 96 well microtitration plates (maxisorp plates; Nunc, Roskilde, Denmark) were BAY 80-6946 coated with 100 μl of LAg (25 μg/ml) diluted in 20 mM phosphate buffer (pH 7.5) overnight at 4°C. Non-specific binding sites were blocked with 1% bovine serum albumin (BSA) in PBS at room GF120918 temperature for 3 h. After washing with PBS containing 0.05% Tween-20 (Sigma-Aldrich),

the plates were incubated overnight at 4°C with 1:1000 dilutions of mice sera. The plates were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG

(Sigma-Aldrich) diluted 1:5000 and antimouse IgG1 or IgG2a (BD Pharmingen, San Diego, USA) diluted 1:1000 in blocking buffer. Finally, colour reaction was developed by the addition of 100 μl/well of substrate solution (o-phenylene diamine dihydrochloride, 0.8 mg/ml in 0.05 M phosphate-citrate buffer, pH 5.0, containing 0.04% H2O2) for 30 min. Absorbance was determined at 450 nm using ELISA plate reader (Thermo, Waltham, USA) [15]. Delayed type hypersensitivity (DTH) After the last vaccination, 2 and 4 months after challenge infection, delayed-type hypersensitivity (DTH) was determined as an index of cell-mediated immunity. The response was evaluated Protein Tyrosine Kinase inhibitor by measuring the difference in the footpad swelling at 24 h following intradermal inoculation of the test footpad with 50 μl of LAg (800 μg/ml) from that of control (PBS- injected) footpad with a constant pressure caliper (Starret, tetracosactide Anthol, USA) [15]. Cytokine Assay Spleens were removed aseptically from experimental mice of each group at 10 days after last immunization and teased between 20 μm pore size sieve into single cell suspension in complete medium prepared with RPMI 1640 supplemented with 10% FBS, 10

mM NaHCO3, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin sulphate, and 50 μM β-mercaptoethanol (Sigma-Aldrich). Erythrocytes were removed by lysis with 0.14 M Tris buffered NH4Cl. The splenocytes were washed twice, resuspended in culture medium and viable mononuclear cell number was determined by Trypan blue exclusion. Splenocytes were then cultured in a 96-well flat-bottomed ELISA plate (Nunc) at a density of 2 × 105 cells/well in a final volume of 200 μl. The cells were restimulated in vitro with medium alone or with LAg (10 μg/ml) and supernatants were collected after 72 h incubation at 37°C in a humified chamber containing 5% CO2 and stored at -70°C until use. Measurements of IFN-γ and IL-4 concentrations were carried out using Opt EIA Kits (BD Pharmingen) as detailed in manufacturers’ instructions [27]. Statistical analysis One-way ANOVA statistical test was performed to assess the differences among various groups.