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Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli
Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong t...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325423/ https://www.ncbi.nlm.nih.gov/pubmed/34395095 http://dx.doi.org/10.7717/peerj.11871 |
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author | Nakamura, Keiji Tokuda, Chikashi Arimitsu, Hideyuki Etoh, Yoshiki Hamasaki, Mitsuhiro Deguchi, Yuichiro Taniguchi, Itsuki Gotoh, Yasuhiro Ogura, Yoshitoshi Hayashi, Tetsuya |
author_facet | Nakamura, Keiji Tokuda, Chikashi Arimitsu, Hideyuki Etoh, Yoshiki Hamasaki, Mitsuhiro Deguchi, Yuichiro Taniguchi, Itsuki Gotoh, Yasuhiro Ogura, Yoshitoshi Hayashi, Tetsuya |
author_sort | Nakamura, Keiji |
collection | PubMed |
description | Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains. |
format | Online Article Text |
id | pubmed-8325423 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-83254232021-08-13 Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli Nakamura, Keiji Tokuda, Chikashi Arimitsu, Hideyuki Etoh, Yoshiki Hamasaki, Mitsuhiro Deguchi, Yuichiro Taniguchi, Itsuki Gotoh, Yasuhiro Ogura, Yoshitoshi Hayashi, Tetsuya PeerJ Biochemistry Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains. PeerJ Inc. 2021-07-28 /pmc/articles/PMC8325423/ /pubmed/34395095 http://dx.doi.org/10.7717/peerj.11871 Text en © 2021 Nakamura et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Biochemistry Nakamura, Keiji Tokuda, Chikashi Arimitsu, Hideyuki Etoh, Yoshiki Hamasaki, Mitsuhiro Deguchi, Yuichiro Taniguchi, Itsuki Gotoh, Yasuhiro Ogura, Yoshitoshi Hayashi, Tetsuya Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli |
title | Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli |
title_full | Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli |
title_fullStr | Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli |
title_full_unstemmed | Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli |
title_short | Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli |
title_sort | development of a homogeneous time-resolved fret (htrf) assay for the quantification of shiga toxin 2 produced by e. coli |
topic | Biochemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325423/ https://www.ncbi.nlm.nih.gov/pubmed/34395095 http://dx.doi.org/10.7717/peerj.11871 |
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