Ex vivo IFN-γ ELISpot assay

The ex vivo IFN-γ ELISpot assay was performed using a human IFN-γ ELISpot kit (capture mAb -D1K; Mabtech). Duplicate wells containing 3 × 10⁵ PBMC were stimulated for 18 h with antigen, PHA as a positive control or media alone as a negative control. Antigens were: a single pool of Ag85A peptides, consisting of 66 15-mer peptides, overlapping by 10 amino acids (2 μg ml⁻¹ per peptide; Peptide Protein Research); BCG from pooled SSI vaccine vials (2 × 10⁵ colony-forming units (CFUs) per ml); PPD from M. tuberculosis (20 μg ml⁻¹; Statens Serum Institute (SSI)); pools of peptides with knows CD8 T-cell epitopes from flu, Epstein–Barr virus (EBV) and CMV peptides (10 μg ml⁻¹ per peptide; Peptide Protein Research); and combined TB10.3 and TB10.4 peptides (10 μg ml⁻¹ per peptide; Peptide Protein Research). Results are reported as SFCs per million PBMCs, calculated by subtracting the mean of the unstimulated wells from the mean of antigen wells and correcting for the numbers of PBMC in the wells. A response was considered positive if the mean number of spots in the antigen well was at least twice the mean of the unstimulated wells and at least five spots greater.