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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

Sensitivity of toxin detection by Vβ-immobilized beads in singleplex assays.Each biotinylated, high-affinity Vβ protein (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC) immobilized on individual streptavidin-coated fluorescent beads was subjected to cognate, toxin binding titration (SEA, SEB, TSST-1, SpeA and SpeC respectively). Toxins captured by the Vβ-immobilized beads were detected with rabbit polyclonal anti-toxin antibodies (anti-SEA, anti-SEB, anti-TSST-1, anti-SEB and anti-SpeC respectively) followed by goat-anti rabbit IgG labeled with Alexa fluor 647. Flow cytometry histograms for each binding titration are shown, with fluorescence arising due to toxin binding on X-axis. Fluorescence in the absence of toxin is represented by gray (filled) trace on each histogram. Median fluorescence units from the histograms were used to generate binding curves, shown on the right. Red, dashed line indicates fluorescence in the absence of toxin. Data shown are representative of experiments performed in triplicate.
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pone.0135986.g003: Sensitivity of toxin detection by Vβ-immobilized beads in singleplex assays.Each biotinylated, high-affinity Vβ protein (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC) immobilized on individual streptavidin-coated fluorescent beads was subjected to cognate, toxin binding titration (SEA, SEB, TSST-1, SpeA and SpeC respectively). Toxins captured by the Vβ-immobilized beads were detected with rabbit polyclonal anti-toxin antibodies (anti-SEA, anti-SEB, anti-TSST-1, anti-SEB and anti-SpeC respectively) followed by goat-anti rabbit IgG labeled with Alexa fluor 647. Flow cytometry histograms for each binding titration are shown, with fluorescence arising due to toxin binding on X-axis. Fluorescence in the absence of toxin is represented by gray (filled) trace on each histogram. Median fluorescence units from the histograms were used to generate binding curves, shown on the right. Red, dashed line indicates fluorescence in the absence of toxin. Data shown are representative of experiments performed in triplicate.

Mentions: In order to determine the specificity and sensitivity of the flow cytometry-based assays using the biotinylated high-affinity Vβ proteins (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC), each Vβ protein was tested in a singleplex assay (Fig 3). Each assay showed a dynamic range of over three orders of magnitude for determining concentrations of the specific toxin. The range spanned the picogram per ml level to hundreds of nanograms/ml level. As determined from the low end of the flow cytometry histograms, the assay detection limits were approximately 400 pg/ml SEA, 3 pg/ml SEB, 25 pg/ml TSST-1, 6000 pg/ml SpeA and 100 pg/ml SpeC (Fig 3).


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)

Sensitivity of toxin detection by Vβ-immobilized beads in singleplex assays.Each biotinylated, high-affinity Vβ protein (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC) immobilized on individual streptavidin-coated fluorescent beads was subjected to cognate, toxin binding titration (SEA, SEB, TSST-1, SpeA and SpeC respectively). Toxins captured by the Vβ-immobilized beads were detected with rabbit polyclonal anti-toxin antibodies (anti-SEA, anti-SEB, anti-TSST-1, anti-SEB and anti-SpeC respectively) followed by goat-anti rabbit IgG labeled with Alexa fluor 647. Flow cytometry histograms for each binding titration are shown, with fluorescence arising due to toxin binding on X-axis. Fluorescence in the absence of toxin is represented by gray (filled) trace on each histogram. Median fluorescence units from the histograms were used to generate binding curves, shown on the right. Red, dashed line indicates fluorescence in the absence of toxin. Data shown are representative of experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135986.g003: Sensitivity of toxin detection by Vβ-immobilized beads in singleplex assays.Each biotinylated, high-affinity Vβ protein (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC) immobilized on individual streptavidin-coated fluorescent beads was subjected to cognate, toxin binding titration (SEA, SEB, TSST-1, SpeA and SpeC respectively). Toxins captured by the Vβ-immobilized beads were detected with rabbit polyclonal anti-toxin antibodies (anti-SEA, anti-SEB, anti-TSST-1, anti-SEB and anti-SpeC respectively) followed by goat-anti rabbit IgG labeled with Alexa fluor 647. Flow cytometry histograms for each binding titration are shown, with fluorescence arising due to toxin binding on X-axis. Fluorescence in the absence of toxin is represented by gray (filled) trace on each histogram. Median fluorescence units from the histograms were used to generate binding curves, shown on the right. Red, dashed line indicates fluorescence in the absence of toxin. Data shown are representative of experiments performed in triplicate.
Mentions: In order to determine the specificity and sensitivity of the flow cytometry-based assays using the biotinylated high-affinity Vβ proteins (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC), each Vβ protein was tested in a singleplex assay (Fig 3). Each assay showed a dynamic range of over three orders of magnitude for determining concentrations of the specific toxin. The range spanned the picogram per ml level to hundreds of nanograms/ml level. As determined from the low end of the flow cytometry histograms, the assay detection limits were approximately 400 pg/ml SEA, 3 pg/ml SEB, 25 pg/ml TSST-1, 6000 pg/ml SpeA and 100 pg/ml SpeC (Fig 3).

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