Humoral response in vaccinated mice following immunization and L. donovani challenge infection. Mice were immunized subcutaneously with PBS, LAg, alum, alum + LAg, saponin, saponin + LAg, or intraperitoneally with Lip and Lip + LAg. ELISA measurement of LAg-specific IgG, IgG1, IgG2a and IgG2b antibodies was performed on sera obtained from mice post-immunization (A), 2 months (B) and 4 months (C) after challenge with L. donovani. The insets in (A) and (C) show antibody levels to the non-leishmanial control antigen OVA. Each sample was examined in duplicate. The results are shown as the mean absorbance values ± SE of five individual mice per group, representative of two independent experiments with similar results. IgG2a/IgG1 ratio in alum + LAg, saponin + LAg and Lip + LAg immunized mice (D) preinfection, 2 months and 4 months postinfection (pi). * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to PBS as well as free adjuvant immunized groups as assessed by one-way ANOVA and Tukey’s multiple comparison test.