There is diversity in the profile of T-cell receptors that are bound by variant SEBs.

<p>We compared the Vβ-TCR expansion in PHA stimulated and SEB variants stimulated PBMC. In all, 24 Vβ-TCRs were analyzed. APC conjugated CD3⁺ blastogenic cells were gated on and simultaneous analysis of 3 Vβ-TCRs per tube was performed. The results are expressed as ratios of the Vβ-TCR expansion induced by SEB from MNHO or SEB from its variants relative to that by PHA with respect to the same individual. Data are shown from two different donor samples. Vβ-TCRs showing a >1 fold increase in ratio of SEB/PHA were considered expanded.</p

Amino acid substitutions in variants of SEB are not predicted to occur in regions that contact immune cell receptors.

<p>A and B are representations of the crystal structure of SEB shown from two different angles. Residues that contact the Vβ-TCR are highlighted in blue and residues that contact MHC II DR1 are highlighted in black. The amino acid substitutions found in variants of SEB are highlighted in red.</p

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

<div><p>Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD₅₀ values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of <i>Staphylococcus aureus</i>. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of <i>Staphylococcus aureus</i> and <i>Streptococcus pyogenes</i>.</p></div

LD₅₀ determination of variant SEBs.

<p>LD₅₀ determination of variant SEBs.</p

Variant SEB superantigenicity is neutralized by a soluble, high-affinity Vβ-TCR protein.

<p>Human PBMCs were exposed to SEB and varying concentrations of the G5-8 high affinity Vβ-TCR protein, and subsequently incubated with ³H-thymidine. Cellular proliferation is reported as the measure of the counts per minute of ³H-thymidine that had incorporated into the DNA of the PBMCs. Error bars represent the standard deviations, and statistical significance was determined by using Student's unpaired <i>t</i>-test. *, <i>P</i><0.05 for statistically significant differences with respect to toxin alone.</p

Summary of sources of <i>Staphylococcus aureus</i> strains analyzed.

<p>^<b>a</b>Sources of the isolates were described by NARSA or ATCC (American Type Culture Collection).</p><p>^<b>b</b>Information about presence or absence of SAg genes was obtained by performing BLAST analysis on genome sequences available in GenBank (NIH genetic sequence database).</p><p>^<b>c</b>Information about presence or absence of SAg genes was obtained as annotations from NARSA website.</p><p>^<b>d</b>Information about presence or absence of SAg genes was established by other investigators (references indicated).</p><p>Where available, information about presence or absence of SAg genes, and/or proteins, is shown. These were also compared with the SAg expression profiles of each strain, as determined by multiplex assay in the present work. “+” represents fluorescence signal above the background in multiplex assay (also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135986#pone.0135986.g006" target="_blank">Fig 6</a>), indicating the presence of toxin indicated.</p

Variant SEBs differ in their ability to cause proliferation of rabbit splenocytes.

<p>Rabbit splenocytes were exposed to varying concentrations of SEB from strains MNBE, MNBD, or MNHO, and subsequently incubated with ³H-thymidine. Cellular proliferation is reported as the measure of the counts per minute of ³H-thymidine that had incorporated into the DNA of the splenocytes. The mitogenicity of each toxin was measured in quadruplicate. The three toxins caused different patterns of splenocyte proliferation. Error bars represent the standard deviations, and statistical significance was determined by using Student's unpaired <i>t</i>-test. *, <i>P</i><0.05 with respect to MNHO. §, <i>P</i><0.05 with respect to MNBE.</p

Multiplex assay in the presence of one toxin.

<p>Solutions containing 50 ng/ml SEA, 10 ng/ml SEB, 10 ng/ml TSST-1, 50 ng/ml SpeA or 10 ng/ml SpeC were tested in multiplex assays. Fluorescence emitted by each Vβ-immobilized bead due to toxin binding, was plotted on bar graphs shown. In all cases, the toxins were detected by the beads immobilized with the high-affinity Vβ engineered for that toxin. The error-bars represent standard deviations from two independent experiments. Similar data (but with higher background) were obtained in other experiments where unoccupied sites on biotin-Vβ immobilized-beads were not blocked with excess biotin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135986#pone.0135986.s004" target="_blank">S4 Fig</a>).</p

Schematic diagram and representative flow cytometry data of the multiplex assay.

<p>Biotinylated, high-affinity Vβ proteins (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC) were immobilized on streptavidin-coated fluorescent beads (P12, P10, P6, P2 and P8 respectively, each having unique fluorescence in the FL-1 channel, λ_emission = 525 nm). For detecting the presence of one or more toxins (SEA and/or SEB in this example), a mixture of Vβ immobilized-beads was added to an unknown sample. Toxins captured by the Vβ-immobilized beads were detected by rabbit polyclonal anti-toxin antibodies followed by goat-anti rabbit IgG labeled with Alexa fluor 647 (λ_emission = 688 nm in FL-4 channel). Scatter plots in the absence or presence of toxin(s) are shown in the lower panel. An increase in fluorescence emitted by the beads in the FL-4 channel (Y-axis) indicates the presence of toxin(s) in the unknown sample. Since each class of beads emits distinct fluorescence in FL-1 channel (X-axis), identity of the toxin present is established by identifying the specific, Vβ-immobilized bead(s) undergoing an increase in fluorescence in the FL-4 channel (Y-axis). In the presence of SEA, only the P12 beads (immobilized with Vβ-SEA) exhibited an increase in fluorescence along Y-axis. In the presence of SEB, P10 beads (immobilized with Vβ-SEB) as well as P2 beads (immobilized with Vβ-SpeA) exhibited increases in fluorescence along Y-axis, due to low affinity of Vβ-SpeA toward SEB. Accordingly, in the presence of SEA and SEB, Vβ-immobilized P12, P10 and P2 beads exhibited increases in fluorescence along Y-axis.</p

Multiplex assay in the presence of a mixture of toxins.

<p>Solutions containing a mixture of two or more toxins were tested in multiplex assays, at the indicated concentrations. Fluorescence emitted by each Vβ-immobilized bead due to toxin binding, was plotted on bar graphs shown. The error-bars represent standard deviations from two independent experiments. Similar data (but with higher background) were obtained in other experiments where unoccupied sites on biotin-Vβ immobilized-beads were not blocked with excess biotin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135986#pone.0135986.s005" target="_blank">S5 Fig</a>).</p