HBsAg-specific antibody responses after intranasal (IN) immunization using large volume. BALB/c mice (n = 5−10) were immunized by a single IN inhalation of 150 µl solution containing 1 µg HBsAg with no adjuvant (none), with 1 µg (pale bars), or 10 µg (black bars), of CT, CTB, LT, LTK63, or CpG ODN; or with CpG ODN combined with CT, CTB, LT, or LTK63 (1 µg each, striped bars). Each bar represents the group geometric mean titer (GMT) of the ELISA end-point dilution titer for HBsAg-specific antibodies [anti-HBs, total immunoglobulin G (IgG)] in plasma taken 4 weeks after immunization. Titers were defined as the highest plasma dilution resulting in an absorbance value two times that of non-immune plasma, with a cut-off value of 0.05. HBsAg, purified hepatitis B surface antigen; CT, cholera toxin; CTB, subunit of cholera toxin; LT, Escherichia coli heat labile enterotoxin; LTK63, nontoxic derivative of Escherichia coli heat labile enterotoxin; CpG ODN, oligodeoxynucleotide containing cytosine-guanine phosphate linked dinucleotides.