Samples (n = 10 mice from each group) were tested in triplicate.
At the end of the incubation, the plates were washed five times with PBS and alkaline phosphatase-conjugated antibodies (goat anti-mouse IgG and goat anti-mouse IgM, dilution 1:2000, 100 μl per well) were added. The plates were incubated for 2 h at room temperature, after than washed with PBS. For detection of spots, 100 μl of BCIP/NBT U0126 substrate was added to each well. Following washing, the plates were left at room temperature to dry. The plates were examined for spots counts using an Axio Imager A1 microscope (Zeiss, Germany). Quantitative evaluation of spots and enumeration of Ig-producing cells was performed via KS ELISPOT 4.10 running under AxioVision software (Zeiss, Germany). Data were evaluated for statistical significance of differences by one-way ANOVA followed by Bonferroni’s multiple comparison tests and Spearman’s rank correlation 3-Methyladenine test.
All data were expressed as mean ± SD. To evaluate the ability of antibodies induced by immunization with M5-BSA and M6-BSA conjugates to react with mannan structure, the specific serum antibodies levels against acid-stable mannan moiety of both C. albicans serotypes and C. guilliermondii after each injection of conjugates were determined (Fig. 2). Detected acid-stable mannan-specific antibodies levels in immune sera were compared with the controls (sera obtained after immunization with saline). M5-BSA conjugate immunization induced increase in mannan-specific IgM levels with maximal peak after the secondary sc booster injection (3rd Ponatinib chemical structure sc) for mannan C. albicans serotype A. Immunization with M5-BSA conjugate induced slight statistically significant increase in mannan-specific IgG for mannan C. albicans serotype A and mannan C. guilliermondii. Nevertheless, mannan-specific IgG levels induced by M5-BSA conjugate immunization did not exceed the levels of mannan-specific IgM levels (Fig. 2). For mannan-specific
IgA levels, we observed no increase using mannan C. albicans serotype A and slight statistically significant increase using mannans of C. albicans serotype B and C. guilliermondii as target antigen. In comparison with M5-BSA conjugate, structurally similar M6-BSA conjugate induced different kinetics of mannan-specific antibodies levels throughout the immunization (Fig. 2). We observed a marked increase in mannan C. albicans serotype A-specific IgM levels after the primary injection (1st) and the primary sc booster injection (2nd) of M6-BSA conjugate followed by significant decrease after the secondary booster injections (both, 3rd sc and 3rd ip administration). Mannan C. albicans serotype B and mannan C.