HBsAg-specific antibody responses after intranasal (IN) immunization using small volume. BALB/c mice (n = 5–10) were immunized by IN inhalation of 10 µg of HBsAg alone (none) or with 1, 10 or 50 µg cholera toxin (CT) and/or CpG-containing oligodeoxynucleotides (CpG) as adjuvants in a volume of 20 µl. Each bar represents the group mean (± standard error of the mean; SEM) of the ELISA end-point dilution titer for HBsAg-specific antibodies (anti-HBs, total immunoglobulin G; IgG) in plasma taken 4 weeks after one (open bars), two (filled bars), or three (striped bars) immunizations. Titers were defined as the highest plasma dilution resulting in an absorbance value two times that of nonimmune plasma, with a cut-off value of 0.05. HBsAg, purified hepatitis B surface antigen; GMT, geometric mean titers.