Luminex cytokine array
Protein levels of IL-2, TNFα, MIP-1β, MCP-1, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13 IL-17A, G-CSF and GM-CSF were measured by commercially available Human Cytokine 17-plex Assays and Human Cytokine Th1/Th2 Assays (Bio-Rad, Hercules, CA, USA), following manufacturer’s instructions. The median fluorescent intensity (MFI) of each cytokine bead set was measured on a Bio-plex 200 instrument (Bio-Rad). Cytokine concentration was calculated from control curves of standards provided in the kit. In the absence of a response to the peptides, the amount of cytokine was considered as below the LLOD. For IL-2, a value of the lowest value on the standard curve was given for that sample (6.28pg/ml). For all other cytokines, this was recorded as out of range. If a response was greater than the highest point on the standard curve, this was also recorded as out of range. As above, cut off for determining a positive response was set by Youden’s index. The IL-2 optimal cut off was determined as >19.91pg/ml in the test for natural infection-induced responses. For the test for vaccine-induced responses, optimal cut off values for IL-2, IL-13 and IL-10 were >37.08pg /ml, >2.28pg/ml and >7.81pg/ml, respectively.