Analysis of antigen-specific immune responses after i.n. immunisation. Balb/c mice were immunised i.n. with 1 μg LT and 10 μg Ag85B-ESAT6, or PBS. (A) Mice were sacrificed 28 days post immunisation and blood collected for evaluation of primary Ag-specific Ab synthesis by ELISA. Titres are shown and reflect five individual mice per group expressed as total Ab titre using a cut-off of OD 0.2 with black bars showing the geometric mean. (B) Spleens were also collected for T cell assays. Cytokine responses were measured upon in vitro stimulation with Ag85B-ESAT6 (5 μg/mL) for 36-48 h. Columns represent the mean ± SD stimulation indices of splenocytes from 5 immunised mice. Statistical significance was determined by using the Mann-Whitney U test (*p < 0.05, **p < 0.01 and ***p < 0.001).