Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout - Fig 4

<p><b>Correlation of serum titers from individual mouse sera against <i>S</i>. Typhimurium (A) and <i>S</i>. Enteritidis (B) as determined by L-SBA and by the conventional SBA assay.</b> Individual mouse sera were simultaneously assayed by L-SBA (y-axis) and by the conventional CFU-based assay (x-axis) against <i>S</i>. Typhimurium (A) and against <i>S</i>. Enteritidis (B). Plots compare titers from each assay and demonstrate a linear correlation with a slope of 1. Black lines represent the fitting by linear regression. SBA titers were obtained by single experiments.</p

Serum titers and standard deviation (SD) values shown in Fig 5.

<p>Serum titers and standard deviation (SD) values shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172163#pone.0172163.g005" target="_blank">Fig 5</a>.</p

Comparison of titers obtained by the conventional SBA assay and by L-SBA, using a panel of standard reference sera or mAbs against different bacterial species.

<p>White or grey bars represent the SBA titers calculated from the conventional CFU-counts or from L-SBA, respectively. Standard reference sera were used against <i>C</i>. <i>freundii</i>, <i>S</i>. Typhimurium, <i>S</i>. Enteritidis, <i>S</i>. <i>flexneri 2a and 3a</i> and <i>S</i>. <i>sonnei</i>. For <i>N</i>. <i>meningitidis</i> A and W anti-capsular mAbs were used. The results are the mean ± standard deviation values from three independent experiments.</p

Comparison of L-SBA and conventional SBA assay with standard reference sera against <i>Shigella flexneri</i> serogroups 2a and 3a.

<p>HI standard reference serum against <i>S</i>. <i>flexneri</i> 2a (A and B) and <i>S</i>. <i>flexneri</i> 3a (C and D) was tested by L-SBA (A and C, respectively) and by the conventional assay (B and D, respectively) in three independent experiments. Black lines represent the curve fitting by nonlinear regression for each replicate.</p

Optimization of bacterial cell concentration for L-SBA.

<p>(A) Different bacterial concentrations were tested with 5% BRS and measured by luminescence at T0 (triangle symbol) and at T180 (square symbol). (B) Bacterial cells at 1.5 x 10⁵ CFU/mL were diluted down until 1.0 x 10³ CFU/mL and measured by luminescence. The area within the dashed lines define the confidence interval of the best-fit line of the linear regression (y = 0.009465x + 10.28). CPS, counts per second.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

L-SBA optimization to circumvent serum interference with luminescence readout.

<p>Bactericidal reaction was tested using <i>C</i>. <i>freundii</i> in the presence of 5% BRS and serial dilutions of HI standard serum. At the end of the SBA reaction, we performed: a. one spin and resuspension of bacterial pellets in PBS; b. one wash; c. two washes in PBS, before measuring luminescence. Average luminescence signals and standard deviations are shown from three independent experiments, for each condition (for some points the error bars are not visible because shorter than the symbols).</p

Serum titers with 95% Confidence Interval (CI) limits from bactericidal reactions shown in Fig 6 against <i>N</i>. <i>meningitidis</i> A (MenA) and <i>N</i>. <i>meningitidis</i> W (MenW).

<p>Serum titers with 95% Confidence Interval (CI) limits from bactericidal reactions shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172163#pone.0172163.g006" target="_blank">Fig 6</a> against <i>N</i>. <i>meningitidis</i> A (MenA) and <i>N</i>. <i>meningitidis</i> W (MenW).</p

SBA titers against <i>S</i>. <i>flexneri</i> 2a and 3a as determined by L-SBA and by CFU counts-based method.

<p>SBA titers against <i>S</i>. <i>flexneri</i> 2a and 3a as determined by L-SBA and by CFU counts-based method.</p

Comparison of L-SBA and conventional assay using pooled mouse sera against <i>Neisseria meningitidis</i> serogroups A and W.

<p>HI pooled mouse sera were tested by L-SBA (A, C) or by conventional assay (B, D) against <i>N</i>. <i>meningitidis</i> A Niga 16/09 strain (A, B) or <i>N</i>. <i>meningitidis</i> W Mali 4/11 strain (C, D). Results are the mean ± standard deviation values from three replicates (for some points the error bars are not visible because shorter than the symbols).</p