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A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies)...

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Autores principales: Gehring, Andrew, He, Xiaohua, Fratamico, Pina, Lee, Joseph, Bagi, Lori, Brewster, Jeffrey, Paoli, George, He, Yiping, Xie, Yanping, Skinner, Craig, Barnett, Charlie, Harris, Douglas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073133/
https://www.ncbi.nlm.nih.gov/pubmed/24921195
http://dx.doi.org/10.3390/toxins6061855
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author Gehring, Andrew
He, Xiaohua
Fratamico, Pina
Lee, Joseph
Bagi, Lori
Brewster, Jeffrey
Paoli, George
He, Yiping
Xie, Yanping
Skinner, Craig
Barnett, Charlie
Harris, Douglas
author_facet Gehring, Andrew
He, Xiaohua
Fratamico, Pina
Lee, Joseph
Bagi, Lori
Brewster, Jeffrey
Paoli, George
He, Yiping
Xie, Yanping
Skinner, Craig
Barnett, Charlie
Harris, Douglas
author_sort Gehring, Andrew
collection PubMed
description Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.
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spelling pubmed-40731332014-06-27 A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins Gehring, Andrew He, Xiaohua Fratamico, Pina Lee, Joseph Bagi, Lori Brewster, Jeffrey Paoli, George He, Yiping Xie, Yanping Skinner, Craig Barnett, Charlie Harris, Douglas Toxins (Basel) Article Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef. MDPI 2014-06-11 /pmc/articles/PMC4073133/ /pubmed/24921195 http://dx.doi.org/10.3390/toxins6061855 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Gehring, Andrew
He, Xiaohua
Fratamico, Pina
Lee, Joseph
Bagi, Lori
Brewster, Jeffrey
Paoli, George
He, Yiping
Xie, Yanping
Skinner, Craig
Barnett, Charlie
Harris, Douglas
A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins
title A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins
title_full A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins
title_fullStr A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins
title_full_unstemmed A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins
title_short A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins
title_sort high-throughput, precipitating colorimetric sandwich elisa microarray for shiga toxins
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073133/
https://www.ncbi.nlm.nih.gov/pubmed/24921195
http://dx.doi.org/10.3390/toxins6061855
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