Lymphocyte proliferation assay.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) dye was used for the proliferation assay of mouse lymphocytes on days 38 and 180 after the first immunization. Mice from each group (n = 4) and at each time point were euthanized under sterile conditions. The spleens were removed, and a single-cell suspension of the spleens was prepared in RPMI 1640 medium (Gibco, Invitrogen, Scotland, UK). Next, by using ammonium chloride-potassium lysis buffer (pH 7.2), the red blood cells were lysed, and the cells were washed. The cells were then resuspended in complete culture medium containing RPMI 1640 medium (Gibco), 5% fetal calf serum (FCS; Sigma-Aldrich), 2.3 × 10⁻² mM 2-mercaptoethanol, penicillin-streptomycin (100 U–100 μg/ml), and 10 mM HEPES (Sigma-Aldrich), and their viability was assessed by trypan blue dye exclusion. Next, 100 μl of the cell suspension (2 × 10⁶ cells/ml) was cultured in a flat-bottom 96-well tissue culture plate (Orange Scientific, EU, Belgium) in triplicates in the presence of concanavalin A (ConA) (5 μg/ml) (as the positive control), PfCelTOS (10 μg/ml), and medium alone (as the negative control). All the cells were incubated in a 5% CO₂ incubator in a humidified atmosphere at 37°C for 48 h. Subsequently, the supernatants were removed, the formazan crystals were dissolved in 0.04 N HCl in absolute isopropanol, and the OD was measured at 550 nm. The stimulation index (SI) was calculated by dividing the mean absorbance of the cells with the antigen by the mean absorbance of the cells without the antigen.