Fig 2.

Enhanced immunogenicity of LAM-CRT and LAM-EGFP over unconjugated laminarin. Groups of BALB/c mice (6 per group) were s.c. injected with laminarin (A), LAM-EGFP (B), or LAM-CRT (C) or with rCRT/39–272 (F) (in PBS; 100 μg/mouse) and boosted with 50 μg of the same Ag preparations 2 weeks later. The mice were bled at different time points thereafter, and the combined sera (1/200 dilution) were assayed, in triplicate wells, for IgM and IgG using laminarin-based ELISAs. The detection antibody was HRP-conjugated goat anti-mouse IgM or IgG with OPD as the substrate. (D) Sera of the LAM-CRT group (mixture of equal proportions, 1/200 dilution), collected on day 28, were assayed in laminarin-based ELISAs for IgG subclasses using HRP-conjugated goat anti-mouse Abs against IgG1, IgG2a, IgG2b, or IgG3 for detection. (E) Antisera from all 3 groups (mixture of equal proportions for each group) were also titrated against laminarin using HRP-labeled IgG1-specific Abs for detection. (F) ELISAs were also carried out to test cross reactivity of antisera from the rCRT/39–272 group using microtiter plates precoated, in triplicate, with rCRT/39–272, rEGFP, pustulan, laminarin, mannan, alginic acid, dextran, and LPS. The results are expressed as mean OD₄₉₂ ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant. Results are representative of three independent experiments.