A globotetraosylceramide (Gb&lt;sub&gt;4&lt;/sub&gt;) receptor-based ELISA for quantitative detection of Shiga toxin 2e

Togashi, Katsuhiro; Sasaki, Shiho; Sato, Wataru

doi:10.1292/jvms.14-0071

Cited by 3 publications

(2 citation statements)

References 18 publications

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“…Under this concentration, we were able to detect Stx2a as low as 23 ng/mL which met our need. In Gb4-based quantitative ELISA for Stx2e, the limit of detection is 20 ng/mL, 38 as comparable as ours. Moreover, two 96 well R-ELISA plates can be prepared from one vial of commercial available CTH (0.5 mg), instead of one 96 well plate if a higher concentration was used.…”

Section: Advances In Food Technology and Nutritional Sciences Issn 23supporting

confidence: 86%

“…First, we did not notice a correlation between Stx2 production and lineage. It was previously reported that E. coli O157:H7 isolates from lineage II produced less Stx2 than those from lineage I and I/II 28 ; however, our findings show that the Stx2 levels in strains from lineage II (PA40, 41) were not significantly different from strains belonging to lineage I (PA5, 31, 32, 49) or lineage I/II (PA22, 35,38,47). Second, although strains from clade 8 were suggested to produce higher Stx2 levels than isolates from other clades, 29,30 our data indicates this is not universal.…”

Section: Advances In Food Technology and Nutritional Sciences Issn 23contrasting

confidence: 80%

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A Quantitative Enzyme-Linked Immunosorbent Assay for Shiga Toxin 2a Requiring Only Commercially Available Reagents

Xiaoli¹,

Dudley²

2017

Adv Food Technol Nutr Sci Open J

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CitationThis study develops a quantitative ELISA for measuring Shiga toxin 2 (Stx2) produced by Shiga toxin (Stx)-producing Escherichia coli (STEC), including foodborne pathogen E. coli O157:H7 by all commercially available reagents. Background: Most foodborne outbreak strains of STEC produce Stx2a, Stx2c or both, which are more frequently associated with human clinical cases, leading to severe gastrointestinal diseases or even death. However, no simple and cheap assays or kits are available for quantitative detection of Stx2a and Stx2c. Therefore, an easy and affordable quantitative method for Stx2 is needed for its pathogenesis study. Results: We successfully developed a sensitive and specific receptor-based ELISA by using all commercial available agents. Hydroxyl acyl ceramide trihexoside, an analogue of Stx2 receptor globotriaosylceramide (Gb3), was used for antigen capture, and several critical steps were identified that must be adhered to ensure repeatability. No cross reactivity was observed with Stx1, and linear curves could be constructed using either purified Stx2a or a bacterial lysate from an Stx2a-producing E. coli O157:H7 strain. We applied this method to quantify Stx2 production by a collection of E. coli O157:H7 strains, indicating it can be extended to qualitatively evaluate Stx2c, and providing evidence that toxin production does not necessarily correlate with strain phylogeny. Conclusion: Our R-ELISA provides a reliable way to quantify Stx2a using commercially available components, and it can also be used for detecting Stx2c. This cost-effective ELISA can be easily performed, suggesting it will be a useful tool for studying pathogenesis of STEC.

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Section: Advances In Food Technology and Nutritional Sciences Issn 23supporting

confidence: 86%

Section: Advances In Food Technology and Nutritional Sciences Issn 23contrasting

confidence: 80%

A Quantitative Enzyme-Linked Immunosorbent Assay for Shiga Toxin 2a Requiring Only Commercially Available Reagents

Xiaoli¹,

Dudley²

2017

Adv Food Technol Nutr Sci Open J

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Detection Methods for Shiga Toxins and Shiga Toxin-Producing E. coli

Silva¹,

Brandon²,

Skinner³

et al. 2017

Shiga Toxins

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Enhanced production of natural shiga toxin by overexpressing A subunit of Stx2e in Stx2e-producing Escherichia coli isolated in South Korea

Hur

Seo

et al. 2022

Preprint

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This study explored the optimal culture conditions for maximizing shiga toxin production in Stx2e-producing Escherichia coli (STEC) 150229, isolated from porcine edema disease (ED), with the goal of preparing a Stx2e toxoid vaccine candidate. High cytotoxicity was observed for this strain [tissue culture cytotoxic dose 50% (10 4 TCCD 50 /100 μl)] from 48 h after incubation. Stx2e was overexpressed by transforming pStx2e A into STEC 150229, resulting in the production of recombinant Stx2e A/B complex combined with intrinsic Stx2e B. The enhanced production of Stx2e was evaluated based on the level of cytotoxicity against Vero cells. The highest cytotoxicity (10 5 TCCD 50 /100 μl) was observed with the samples of recombinant Stx2e A/B complex eluted with 500 mM imidazole at 48 h of incubation. In conclusion, the recombinant Stx2e A protein forms an active protein complex with the intrinsic Stx2e B component from STEC 150229, producing high levels of shiga toxin.

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A globotetraosylceramide (Gb<sub>4</sub>) receptor-based ELISA for quantitative detection of Shiga toxin 2e

Cited by 3 publications

References 18 publications

A Quantitative Enzyme-Linked Immunosorbent Assay for Shiga Toxin 2a Requiring Only Commercially Available Reagents

A Quantitative Enzyme-Linked Immunosorbent Assay for Shiga Toxin 2a Requiring Only Commercially Available Reagents

Detection Methods for Shiga Toxins and Shiga Toxin-Producing E. coli

Enhanced production of natural shiga toxin by overexpressing A subunit of Stx2e in Stx2e-producing Escherichia coli isolated in South Korea

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