Experimental design for simultaneous detection of anti-Trypanosomatidae IgG1 antibodies by flow cytometry-based triplex array (FC-TRIPLEX Chagas/Leish IgG1).

<p>(A) Major assay steps. PBS, phosphate-buffered saline; FBS, fetal bovine serum; PE, phycoerythrin. (B) Parasite gating strategy based on FCS versus SSC dot plot distribution followed by a set marker setup for internal control positivity limit, leading to a Percentage of Positive Fluorescent Parasites value of <2.0%. (C) This set marker setup was maintained to subsequently determine disease-related reactivity patterns. VL, visceral leishmaniasis; CH, Chagas disease; LCL, localized cutaneous leishmaniasis; NI, noninfected controls. Dark frames highlight species-specific IgG1 reactivity patterns at selected serum dilutions (VL, anti-<i>L</i>. <i>infantum</i> at 1:32,000; CH, anti-<i>T</i>. <i>cruzi</i> at 1:2,000; LCL and NI, anti-<i>L</i>. <i>braziliensis</i> at 1:1,000) according to Garcia et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref013" target="_blank">13</a>], Matos et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref015" target="_blank">15</a>], and Pereira et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122938#pone.0122938.ref012" target="_blank">12</a>], respectively.