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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

Expression and biotinylation of high-affinity Vβ proteins.(A) Purification of monomeric fractions of refolded, high-affinity Vβ proteins by size-exclusion chromatography. Dashed line indicates the molecular weight standards. (B) Gel-shift assay for monitoring biotinylation of high-affinity Vβ proteins (Vβ). Disappearance of biotinylated-Vβ (bVβ) (~15kDa band) in the presence of streptavidin (SAv) was indicative of biotinylation.
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pone.0135986.g002: Expression and biotinylation of high-affinity Vβ proteins.(A) Purification of monomeric fractions of refolded, high-affinity Vβ proteins by size-exclusion chromatography. Dashed line indicates the molecular weight standards. (B) Gel-shift assay for monitoring biotinylation of high-affinity Vβ proteins (Vβ). Disappearance of biotinylated-Vβ (bVβ) (~15kDa band) in the presence of streptavidin (SAv) was indicative of biotinylation.

Mentions: In order to generate bead-based arrays of high-affinity capture agents, the genes encoding five high-affinity Vβ proteins were cloned with an N-terminal 6X-His tag for purification and a C-terminal ‘Avitag’ sequence (GGGLNDIFEAQKIEWHE) for enzymatic linkage of biotin to lysine. The proteins were expressed in E. coli and purified from inclusion bodies after in vitro refolding. Ni-affinity purified proteins were subjected to S200 gel filtration yielding a substantial fraction of monomeric Vβ protein in each case (~15 to 18kDa apparent mobility) (Fig 2A). The monomeric fractions were concentrated and biotinylated in vitro by addition of BirA ligase and 100 μM biotin.


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)

Expression and biotinylation of high-affinity Vβ proteins.(A) Purification of monomeric fractions of refolded, high-affinity Vβ proteins by size-exclusion chromatography. Dashed line indicates the molecular weight standards. (B) Gel-shift assay for monitoring biotinylation of high-affinity Vβ proteins (Vβ). Disappearance of biotinylated-Vβ (bVβ) (~15kDa band) in the presence of streptavidin (SAv) was indicative of biotinylation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135986.g002: Expression and biotinylation of high-affinity Vβ proteins.(A) Purification of monomeric fractions of refolded, high-affinity Vβ proteins by size-exclusion chromatography. Dashed line indicates the molecular weight standards. (B) Gel-shift assay for monitoring biotinylation of high-affinity Vβ proteins (Vβ). Disappearance of biotinylated-Vβ (bVβ) (~15kDa band) in the presence of streptavidin (SAv) was indicative of biotinylation.
Mentions: In order to generate bead-based arrays of high-affinity capture agents, the genes encoding five high-affinity Vβ proteins were cloned with an N-terminal 6X-His tag for purification and a C-terminal ‘Avitag’ sequence (GGGLNDIFEAQKIEWHE) for enzymatic linkage of biotin to lysine. The proteins were expressed in E. coli and purified from inclusion bodies after in vitro refolding. Ni-affinity purified proteins were subjected to S200 gel filtration yielding a substantial fraction of monomeric Vβ protein in each case (~15 to 18kDa apparent mobility) (Fig 2A). The monomeric fractions were concentrated and biotinylated in vitro by addition of BirA ligase and 100 μM biotin.

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