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A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

Sharma P, Wang N, Chervin AS, Quinn CL, Stone JD, Kranz DM - PLoS ONE (2015)

Bottom Line: 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.Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Illinois, Urbana, Illinois, United States of America.

ABSTRACT
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 Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

No MeSH data available.


Related in: MedlinePlus

Multiplex assay in the presence of one toxin.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 (S4 Fig).
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pone.0135986.g004: Multiplex assay in the presence of one toxin.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 (S4 Fig).

Mentions: In order to detect and quantitate multiple toxins in the same sample, a mixture of Vβ-immobilized beads were incubated with specific toxins and bound toxins were detected with a mixture of the polyclonal anti-toxin antibodies (Fig 4). In this assay format, SEA, SEB, TSST-1, SpeA and SpeC were detected by their Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC probes, respectively (Fig 4). As expected, the Vβ-SpeA exhibited some cross-reactivity with SEB at concentrations higher than 10 ng/ml (S3 Fig); [34]. The Vβ-TSST-1 also cross-reacted with SpeC at concentrations higher than 1 ng/ml (S3 Fig), probably because TSST-1 and SpeC both bind to the same human Vβ region, Vβ2.1, and in fact the Vβ-TSST-1 and Vβ-SpeC high-affinity mutants were both engineered from the human Vβ2.1 mutant EP-8 that binds to both SAgs [32]. Similarly, we also observed that Vβ-SpeC cross-reacted with TSST-1 at concentrations higher than 10 ng/ml.


A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

Sharma P, Wang N, Chervin AS, Quinn CL, Stone JD, Kranz DM - PLoS ONE (2015)

Multiplex assay in the presence of one toxin.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 (S4 Fig).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549143&req=5

pone.0135986.g004: Multiplex assay in the presence of one toxin.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 (S4 Fig).
Mentions: In order to detect and quantitate multiple toxins in the same sample, a mixture of Vβ-immobilized beads were incubated with specific toxins and bound toxins were detected with a mixture of the polyclonal anti-toxin antibodies (Fig 4). In this assay format, SEA, SEB, TSST-1, SpeA and SpeC were detected by their Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC probes, respectively (Fig 4). As expected, the Vβ-SpeA exhibited some cross-reactivity with SEB at concentrations higher than 10 ng/ml (S3 Fig); [34]. The Vβ-TSST-1 also cross-reacted with SpeC at concentrations higher than 1 ng/ml (S3 Fig), probably because TSST-1 and SpeC both bind to the same human Vβ region, Vβ2.1, and in fact the Vβ-TSST-1 and Vβ-SpeC high-affinity mutants were both engineered from the human Vβ2.1 mutant EP-8 that binds to both SAgs [32]. Similarly, we also observed that Vβ-SpeC cross-reacted with TSST-1 at concentrations higher than 10 ng/ml.

Bottom Line: 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.Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Illinois, Urbana, Illinois, United States of America.

ABSTRACT
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 Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

No MeSH data available.


Related in: MedlinePlus