b-galactosidase-specific antibody responses were determined by using an enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA plates (high binding capacity, Greiner, Alphen a/d Rijn, the Netherlands) were coated overnight at ambient temperature with b-galactosidase (100 ng in 100 ll/well) in coating buffer (0.05 M carbonate/bicarbonate, pH 9.6). To measure antibody responses in mice vaccinated with liposomes containing expressed b-galactosidase, the ELISA plates were coated with recombinantly produced b-galactosidase in E. coli strain BL21 (DE3) (Invitrogen Carlsbad, CA) and purified by using an Akta Purifier equipped with 5 mL His-Trap HP columns (GE Healthcare, Uppsala, Sweden). For serum samples of mice vaccinated with control formulations containing b-galactosidase encapsulated in liposomes, the plates were coated with the same commercial source of b-galactosidase that was used for entrapment. After coating, plates were washed and blocked by incubation with 2.5% (w/v) milk powder in coating buffer (200 ll/well) for 1 h at 37°C. Subsequently, the plates were washed with PBS containing 0.05% Tween, pH 7.4 (PBS/Tween). Appropriate dilutions of sera of each individual mouse were applied to the plates, serially diluted two-fold in PBS/ Tween and incubated for 2 h at 37°C. Plates were then washed and incubated either with horseradish peroxidase (HRP)-conjugated goat antibody against mice IgG or HRPconjugated rabbit antibody against mice IgG1 or HRPconjugated rat antibody against mice IgG2a (all diluted 1:5000 in PBS/Tween, 100 ll/well) for 1 h at 37°C. Thereafter, the plates were washed twice with PBS/Tween and once with PBS. Specific antibodies were detected by adding 100 ll of 3,3 0 ,5,5 0 -tetra methyl benzidine (TMB, 0.1 mg/ml) in 10 mM sodium acetate pH 5.5 buffer also containing 0.06% (v/v) hydrogen peroxide to each well. After 10 min, the reaction was stopped by adding 50 ll 2 M H 2 SO 4 to each well. Total IgG, IgG1 and IgG2a antibody titers are expressed as the reciprocal sample dilution corresponding with an A 450 of 0.2 above the background (Amidi et al. 2007 ). Comparison between mice of different groups with positive titers was made by a oneway ANOVA test.