An Improved Method for the Sensitive Detection of Shiga Toxin 2 in Human Serum

He, Xiaohua; Ardissino, Gianluigi; Patfield, Stephanie; Cheng, Luisa W.; Silva, Christopher J.; Brigotti, Maurizio

doi:10.3390/toxins10020059

Cited by 14 publications

(15 citation statements)

References 26 publications

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“…Negligible amounts of free Stx have been found in the sera of patients with HUS [45]; the detection of Stx2 may have been hampered by the presence in the human serum of Stx2-binding components, including serum amyloid P component. A new improved method may have overcome this problem, enabling early and sensitive detection of Stx2 in the sera of STEC-infected patients, so that preventive measures can be adopted in a timely manner [46]. Stx has been detected in the sera bound to blood cells or in blood cell-derived microvesicles.…”

Section: Pathogenetic Mechanisms Of Stec-husmentioning

confidence: 99%

Complement Activation Contributes to the Pathophysiology of Shiga Toxin-Associated Hemolytic Uremic Syndrome

et al. 2019

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Shiga toxin (Stx)-producing Escherichia coli (STEC) infections have become a threat to public health globally because of the severe illnesses that they can trigger, such as hemorrhagic colitis and the post-diarrheal hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. Glomerular endothelial cells are primary targets of Stx which, after binding to its specific receptor globotriaosylceramide, upregulates proinflammatory proteins involved both in the recruitment and adhesion of leukocytes and thrombus formation at the site of endothelial injury. In this review, we discuss the role of complement activation in promoting glomerular microvascular dysfunction, providing evidence from experimental models and patients with STEC-HUS. Within the glomerulus, an important target for Stx-induced complement activation is the podocyte, a cell type that is in close contact with endothelial cells and participates in maintaining the filtration barrier. Recently, podocyte injury and loss have been indicated as potential risk factors for long-term renal sequelae in patients with STEC-HUS. Therapeutic approaches targeting the complement system, that may be useful options for patients with STEC-HUS, will also be discussed.

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Section: Pathogenetic Mechanisms Of Stec-husmentioning

confidence: 99%

Complement Activation Contributes to the Pathophysiology of Shiga Toxin-Associated Hemolytic Uremic Syndrome

et al. 2019

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“…The proposed method might be useful for the detection of cell-free Stx2a which circulate in human blood. Free Stx2a was found in sera from STEC-infected patients before the onset of eHUS [4,17,18] and its cleaved form could be easily detected by this method. The other form of cell-free Stx2a found in patients's sera is particulate toxin (i.e., associated to extracellular vesicles), which is strictly related to the development of eHUS [4].…”

Section: Discussionmentioning

confidence: 94%

“…Concentrated human serum did not induce any effect on translation, although partially inhibiting protein synthesis in the absence of DTT. Therefore, we advise the measurement of Stx2a concentrations in patients' serum by ELISA [17], for a comparison to be made between the obtained value (ng/mL) with the range of concentrations detectable by our method (Scheme II). If no inhibition is predicted, the serum sample should be assayed in the presence and in the absence of DTT to confirm this prediction and rule out the presence of unspecific Stx2a-unrelated effects on protein synthesis.…”

Section: Lowering the Detection Limit Of The Cell-free Translation Symentioning

confidence: 99%

Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum

Rocchetti

Munari

Varrone

et al. 2021

Toxins

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The pathogenesis of Escherichia coli-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing E. coli (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum proteins) whose interaction is crucial for the immediate outcome and for the development of this life-threatening sequela. Stx2a, associated to circulating cells (early toxemia) or extracellular vesicles (late toxemia) in blood, is considered the main pathogenic factor in the development of eHUS. Recently, it was found that the functional properties of Stx2a (binding to circulating cells and complement components) change according to modifications of the structure of the toxin, i.e., after a single cleavage of the A subunit resulting in two fragments, A1 and A2, linked by a disulfide bridge. Herein, we describe a method to be used for the detection of the cleaved form of Stx2a in the serum of STEC-infected or eHUS patients. The method is based on the detection of the boosted inhibitory activity of the cleaved toxin, upon treatment with reducing agents, on a rabbit cell-free translation system reconstituted with human ribosomes. The method overcomes the technical problem caused by the presence of inhibitors of translation in human serum that have been stalled by the addition of RNAase blockers and by treatment with immobilized protein G. This method, allowing the detection of Stx2a at concentrations similar to those found by ELISA in the blood of STEC-infected patients, could be a useful tool to study the contribution of the cleaved form of Stx2a in the pathogenesis of eHUS.

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“…Other potential benefits may also be realized such as the fact that the AlphaLISA requires the toxin to be intact since the signal is dependent upon the A and B subunits being within a set distance to one another. This AlphaLISA might also be applicable for quantifying the amount of Stx2 in other matrices, indicating a use not only for detecting the presence of STECs in food as presented here but for use in clinical samples consisting of serum/stool [ 30 , 31 ] or for research investigating variables related to stx induction as well [ 32 ].…”

Section: Discussionmentioning

confidence: 99%

Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

Armstrong

Ruth

Capobianco

et al. 2018

Toxins

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Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.

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An Improved Method for the Sensitive Detection of Shiga Toxin 2 in Human Serum

Cited by 14 publications

References 26 publications

Complement Activation Contributes to the Pathophysiology of Shiga Toxin-Associated Hemolytic Uremic Syndrome

Complement Activation Contributes to the Pathophysiology of Shiga Toxin-Associated Hemolytic Uremic Syndrome

Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum

Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

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