The development of diagnostic tests to differentiate between vaccinated animals and those infected with Mycobacterium bovis is required so that test and slaughter control strategies can continue alongside vaccination. In this work, the peptide antigen, ESAT-6, p45, derived from the N-terminal sequence of the ESAT-6 protein, was adsorbed onto a range of microparticulate and nanoparticulate substrates to enhance the in vitro immune response of blood lymphocytes previously sensitised to M. bovis. Two types of hydroxyapatite (HA) nanoparticles (both ∼300 nm in linear dimension), carbonate hydroxyapatite nanospheres (CHA, ∼50 nm), two sizes of polystyrene nanospheres (∼500 and 40 nm), calcium carbonate microparticles (0.3-1.0 μm) and glass microspheres (1.0-3.0 μm) were incubated in a solution of the peptide in PBS. Peptide adsorption increased on the nanoparticle carriers in the order HA (2.5±0.12%w/w), CHA (4.9±0.12) polystyrene (500 nm, 6.8±0.15%, 40 nm, 9.2±0.07) and these systems exhibited fairly low levels of desorption (approximately 10-15% peptide release) over a 24-h incubation period in PBS at 37°C. HA, CHA and polystyrene carriers with adsorbed peptide were subsequently tested in the BOVIGAM™ assay to investigate the efficiency of the immune response of blood lymphocytes in terms of interferon-γ (IFN-γ) production. A general elevation of IFN-γ production resulted for particle-bound peptide relative to free peptide at high peptide concentrations (>10 μg/ml). Only HA-adsorbed peptide resulted in consistently higher immune responses at low peptide concentration (<0.1 μg/ml) compared with the free peptide, indicating that peptide antigens adsorbed to hydroxyapatite nanoparticles may be useful, in diagnostic assays, for differentiating between tuberculosis (TB)-infected and vaccinated animals.