Positive controls were purchased and quantified
and included on each plate. Log-transformed values of test samples were analyzed using linear regression and compared to a standard curve. Samples for a single subject obtained at several time-points were Verteporfin datasheet tested on the same ELISA plate. ELISA plates (Nunc Maxisorp) were coated using rPA (1 μg/mL) for 2–5 days at 4 °C. Test samples diluted into phosphate buffered saline (PBS) that contained 5% milk powder (DIFCO Laboratories, Detroit, MI) and 0.05% Tween 20 (PBSMT) were added and incubated for 1 hour at 37 °C. Plates were washed using PBS with 0.5% Tween-20 (PBST), HRP anti-human IgG (Kirkegaard and Perry Laboratories (KPL); Gaithersburg, MD) added, and incubated for 1 hour at 37 °C, washed using PBST and HDAC inhibitor review developed using ABTS colorigenic substrate (KPL). Data were analyzed using a 4-parameter logistic fit, compared to Emergent’s reference antiserum that was qualified at Battelle Eastern Science and Technology (lot # BEST RS.EBS.001). For ELISpot analysis, PBMC samples were available for 94 subjects. ELISpot subjects were excluded that failed positive control stimulant cut-offs defined as a minimum of 15 CEF I SFC or 200 PHA SFC. Empirical definition of an antigen-specific positive response (for subjects not excluded per above criteria) was set at a minimum of 9 SFC in wells with rPA (or PAp) and at least two-fold higher than background (SFC counts in wells with
medium alone). Scharp analysis  calculated the positive responder rates to PAp and rPA, using triplicate SFC counts entered online http://www.scharp.org/zoe/runDFR/. Scharp analyses are based on distribution-free random sampling (DFR) to increase the strength of the analysis. Those samples having ELISpot data for medium alone (negative control), PAp and rPA were included in the analysis for the Scharp analysis requirement of at least three treatments, aminophylline tested in three or more replicates. The Suissa-Shuster Exact test  was performed to compare the response rate due to different dose levels of AVA and AV7909. IP-10 and IL-6
results were analyzed by a General Linear model with post hoc analysis using MANOVA. The Spearman’s rank correlation coefficient method was used to measure associations between biomarkers. The time course of IP-10 and IL-6 serum levels in AV7909 recipients increased over 24–48 h in a manner consistent with that previously reported  with peak serum levels observed at 24 h, as shown in Fig. 1 and Fig. 2. Post hoc analysis (by group) for IP-10, revealed that all AV7909 groups were statistically different from AVA and saline (placebo) groups. Post hoc analysis for IL-6 (by group) revealed a trend toward higher IL-6 for AV7909 than AVA that was not statistically different, yet both were statistically different from the saline group (Fig. 2). Like IP-10, IL-6 serum levels returned to pre-immunization levels by day 7.