Receiver Operating Characteristics (ROC) plots of the ELISAs of Hcp1, OPS and combined Hcp1/OPS with different groups of serum.

<p>(A) Serum form melioidosis patients versus Thai healthy donors, (B) Serum form melioidosis patients versus U.S. healthy donors. The serum samples were diluted 1:2000 for all ELISAs.</p

Antibody titers to Hcp1 (A) and OPS (B) in diabetic and non-diabetic melioidosis patients.

<p>ELISAs were performed on the plates coated with antigens using undiluted sera and two-fold serially diluted sera from patients. Box plots represent 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. The plots show antibody titers for each group of patients. The antibody titer was determined by ELISA using cut-off titers at specificity of 95%.</p

Antibodies to whole cell antigens of <i>B</i>. <i>pseudomallei</i> K96243 wild type and OPS mutant (<i>ΔwbiD</i> K96243).

<p>ELISAs were performed on the plates coated with antigens using serum from melioidosis patients, Thai healthy donors and U.S. healthy donors at dilution of 1:2000. Box plots represent 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. The plots show OD 450 nm for each antigen.</p

Antibody titers to Hcp1 antigen (A) and OPS antigen (B) in melioidosis patients who survived or died.

<p>ELISAs were performed on the plates coated with antigens using undiluted sera and two-fold serially diluted sera from patients. Box plots represent 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. The plots show antibody titers for each group of patients. The antibody titer was determined by ELISA using cut-off titers at specificity of 95%.</p

NF-κB activation by rFliC.

<p>(A) HEK-Blue^TM-hTLR5 cells (1.4 x 10⁵cells/ml) in HEK-Blue™ Detection medium were incubated with 1, 10, and 100 ng/ml rFliC in three independent experiments. The cells stimulated with flagellin purified from <i>S</i>. Typhimurium (FLA-ST) were used for comparison. After incubation for 24 h, NF-κB activation was determined by monitoring SEAP production. (B) THP1-Dual^TM cells (5.6 x10⁵cells/ml) were added into a 96-well plate containing 20 μl of rFliC at indicated concentrations. The supernatant was collected at 24 h after incubation and SEAP activity was measured using the Quanti-Blue assay. The result of THP-1 cell assay was obtained from three independent experiments. Data represent the mean, and error bars represent the standard deviation of the results of three independent experiments conducted in triplicate.</p

Scatterplots of individual antibody titers to Hcp1 and OPS in serum samples of 103 melioidosis patients who were survived within one year.

<p>(A) Sera from all time points, (B) sera of week 0, (C) sera of week 12 and (D) sera of week 52. For each plot, the relationship between variables as determined by linear regression (dotted line), Spearman correlation (correlation coefficient, rho) and P-value are shown.</p

Sensitivity and specificity of Hcp1-ELISA, OPS-ELISA and Hcp1/OPS-ELISA.

<p>The assay values were calculated from Thai patients who had melioidosis, tuberculosis, scrub typhus, leptospirosis and Thai healthy donors. The cut-off values with Thai healthy donors as controls were used at specificity of 95% for each assay.</p

ELISA results of flagellin-specific IgM and IgG antibodies.

<p>ELISAs were evaluated for IgM and IgG antibodies using sera from melioidosis patients (N = 45) and healthy donors (N = 45) on the pre-coated plate with 15 μg/ml of rFliC. Box plots show OD at 450 nm of rFliC-specific IgG (A) and IgM (B) in different groups of subjects. All data in box plots are presented as 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. (C) Receiver Operating Characteristics (ROC) plots.</p

Differential cytokine profiles from individual subjects.

<p>Whole blood from healthy subjects (N = 14) was stimulated with rFliC at a final concentration of 500 ng/ml. The supernatants were collected at 6 h and 24 h after incubation and pro-inflammatory cytokine (IL-1β, IL-6 and TNF-α) productions were evaluated by ELISA. Each line represents an individual subject. The differences of medians between 6h and 24h were tested by the Mann-Whitney test.</p

Humoral responses to rFliC.

<p>ELISAs using HRP-conjugated rabbit anti-human IgM (A-C) and IgG (D-F) were conducted on prepared plates coated with 15 μg/ml of rFliC. Serum samples from 200 melioidosis patients were used at a dilution of 1:300. Box plots show OD at 450 nm for different groups of patients. All data in box plots are presented as 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. Mann–Whitney test was used to assess for statistically significant difference of median OD values between different serum groups.</p