Quantifying Antibody and Cytokine Responses

Three mice from each immunization group were sacrificed on day 14 after treatment, and serum were separated from whole blood. Antibodies against C. burnetii WCA, recombinant proteins, or peptides were measured by ELISA as described previously [24] (link). Briefly, 100 µl of antigen solution (2 µg/ml) was applied to each well to coat the microplates (Corning, Corning, NY), and each serum sample was diluted 1∶50. The optical density (OD) of each well was read at 450 nm using a UVM340 microplate reader. A multiplex immunoassay in a Luminex Bio-Plex 200 IS 100 instrument (BIO-RAD, Hercules, CA) was also used to quantify serum cytokines including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), IFN-γ, and TNF-α. All measurements were performed in at least triplicate, as described previously [24] (link), [25] (link). Multiplex kits and related reagents were purchased from Affymetrix (Santa Clara, CA).