Heparinised blood samples from all groups of animals were stored overnight at room temperature before stimulation (dos50 µl in duplicate wells of a 96-well plate) for 20 to 24 h at 37 °C in 5% CO2 with MDT and DST reagents at a final assay concentration of 0.1 µg/ml for each protein/peptide component. As positive and negative controls, blood samples were cultured with pokeweed mitogen (10 µg/ml; Sigma, UK) and RPMI-1640 alone (Gibco, UK) respectively. After stimulation, blood was centrifuged at 300 g for 10 min and the plasma supernatant was harvested and stored at ? 80 °C until required.