Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E

Rasooly, Reuven; Paula, María; Hernlem, Bradley J.

doi:10.3390/toxins8050150

Cited by 8 publications

(8 citation statements)

References 37 publications

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“…We used an established cellular model for assessment of PD-1 inhibitory function 13 and stimulated the transfected primary T cells with Raji cells stably expressing human PD-L1 ( Supplementary Fig. 11) with or without prior loading with SEE 40,41 . Co-culture with Raji-PD-L1 loaded with SEE but not without SEE resulted in an elevated luminescent signal that was increased by transfection with SHP-2 WT (Fig.…”

Section: Resultsmentioning

confidence: 99%

Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation

et al. 2020

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Programmed cell death-1 (PD-1) inhibits T cell responses. This function relies on interaction with SHP-2. PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM) at Y223 and one immunoreceptor tyrosine-based switch motif (ITSM) at Y248. Only ITSM-Y248 is indispensable for PD-1-mediated inhibitory function but how SHP-2 enzymatic activation is mechanistically regulated by one PD-1 phosphotyrosine remains a puzzle. We found that after PD-1 phosphorylation, SHP-2 can bridge phosphorylated ITSM-Y248 residues on two PD-1 molecules via its amino terminal (N)-SH2 and carboxyterminal (C)-SH2 domains forming a PD-1: PD-1 dimer in live cells. The biophysical ability of SHP-2 to interact with two ITSM-pY248 residues was documented by isothermal titration calorimetry. SHP-2 interaction with two ITSM-pY248 phosphopeptides induced robust enzymatic activation. Our results unravel a mechanism of PD-1: SHP-2 interaction that depends only on ITSM-Y248 and explain how a single docking site within the PD-1 cytoplasmic tail can activate SHP-2 and PD-1-mediated inhibitory function.

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Section: Resultsmentioning

confidence: 99%

Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation

et al. 2020

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“…It is possible that the observed difference in T-cell responses between SEA and SEE is the binding specificities to the TCR (T-cell receptor) β chain protein expressed on the CCRF-CEM T-cell line. When a Jurkat T-cell line was used in a similar cell-based assay for SEE, that assay was very specific to SEE with no cross reactivity to SEA [14,15]. SEA food poisoning is often associated with milk, but whole milk interferes with and reduces cytokine secretion and significantly reduced SEA detection (Figure 5).…”

Section: Discussionmentioning

confidence: 99%

“…Both CCRF-CEM and Raji cells were maintained in cell culture medium. Murine splenocytes were isolated from spleens aseptically as previously described [14]. All cells were maintained in a 37 °C incubator under a humidified 5% CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

“…Also, antibodies against SEA can react with food matrices to give false-positive results [13]. Cell-based toxin active assays have been developed for detection and quantification of the related Staphylococcal enterotoxin type E (SEE) [14,15]. Jurkat T-cells were used in those assays for SEE and secretion of the cytokine interleukin-2 (IL-2) by Jurkat cells were used for the specific detection of that SE subtype [16].…”

Section: Introductionmentioning

confidence: 99%

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Alternative to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type A

Rasooly

Paula

et al. 2018

Toxins

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Staphylococcal enterotoxins (SEs) are a food safety concern. Existing methods for biologically active SE detection rely on the emetic response in live kittens or monkeys. This method suffers from low sensitivity, poor reproducibility, and causes ethical concerns regarding the use of experimental animals. The Lautenberg Chemical Safety Act encourages the development and adoption of alternatives to testing on animals for chemical toxicity methodologies. In this study, we utilized the superantigenic effect of SE type A (SEA) and used an ex vivo bioassay as an alternative to live animal testing. We found that interleukin-2 (IL-2) secreted by splenocyte can be utilized for quantifiable detection of SEA in food products. To avoid food matrix interference and attenuation of signal, we separated SEA from spiked food products by employing immunomagnetic beads that were coated with an anti-SEA antibody. This ex vivo method has achieved the detection of 1 ng mL−1 of SEA, which is 107 times more sensitive than the existing live animal testing methods. However, this ex vivo bioassay requires sacrificing of mice. To overcome this limitation, we established a cell based in vitro assay using CCRF-CEM, a human CD4+ T-cell line, for the quantitative detection of SEA. Incubation of SEA with CCRF-CEM human T-cells and Raji cells led to quantifiable and dose dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEA, compared with the related SE toxin subtypes B, D, and E or heat inactivated SEA, which produce no secretion of IL-2. This is the first demonstration of an alternative assay that completely eliminates the use of animals for quantitative detection of active SEA.

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“…Staphylococcal enterotoxins (SEs) are the second most common causal agents of food poisoning and clinical infections throughout the world [1,2]. Therefore, its paramount importance to use quick, accurate and sensitive methods for detecting them.…”

Section: Introductionmentioning

confidence: 99%

Cloning, Sequencing and Production of Recombinant Polyclonal Antibodies against Egyptian Staphylococcal Enterotoxin A

El-Din¹,

el-naga²,

El-Fouly³

et al. 2017

AJMR

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Staphylococcus aureus (S.aureus) is the most common pathogen found in hospitals, community-acquired infections and colonizes up to 50% of humans, including mucous membranes and damaged skin. Some strains produce enterotoxins (Ent) as staphylococcal enterotoxin A (SEA) which is involved in 75% of food poisoning outbreaks. Few methods are sensitive and specific enough to confirm the diagnosis of staphylococcal food poisoning. In this study, a segment of Ent A-ORF with molecular weight 774 bp was amplified, cloned, sequenced and aligned with published Ent A-ORFs. Ent A-ORF was subcloned into bacterial expression vector (GST-PGEX4T1 vector) and expressed as a fusion protein with GST-tagged protein. A band size of 27 kDa of purified Ent A protein was cleaved from GST protein by thrombin. The expressed protein of Ent A was identified by strong reaction with commercialized polyclonal antibodies against Ent A. Antiserum against Egyptian recombinant Ent A protein was produced by immunization of Balb/c mice, the produced recombinant polyclonal antibodies had a titre of 1:2500 in direct ELISA. The produced recombinant polyclonal antibody was evaluated and reacted specifically in Western immunoblotting analysis and ELISA test. Finally from the obtained results, the produced recombinant protein of Ent A can be used successfully for production of recombinant polyclonal antibodies and used in large-scale detection of staphylococcal enterotoxin A.

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Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E

Cited by 8 publications

References 37 publications

Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation

Interaction of SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation

Alternative to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type A

Cloning, Sequencing and Production of Recombinant Polyclonal Antibodies against Egyptian Staphylococcal Enterotoxin A

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