Toxins

Research

Jump to: Review

1397 KiB

Open AccessArticle

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

by Andrew Gehring, Xiaohua He, Pina Fratamico, Joseph Lee, Lori Bagi, Jeffrey Brewster, George Paoli, Yiping He, Yanping Xie, Craig Skinner, Charlie Barnett and Douglas Harris

Toxins 2014, 6(6), 1855-1872; https://doi.org/10.3390/toxins6061855 - 11 Jun 2014

Cited by 9 | Viewed by 7021

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

343 KiB

Open AccessArticle

Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates

by Letícia B. Rocha, Christiane Y. Ozaki, Denise S. P. Q. Horton, Caroline A. Menezes, Anderson Silva, Irene Fernandes, Fabio C. Magnoli, Tania M. I. Vaz, Beatriz E. C. Guth and Roxane M. F. Piazza

Toxins 2013, 5(12), 2384-2402; https://doi.org/10.3390/toxins5122384 - 2 Dec 2013

Cited by 16 | Viewed by 7777

Abstract

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no [...] Read more.

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

496 KiB

Open AccessArticle

A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

by Larry H. Stanker, Miles C. Scotcher, Luisa Cheng, Kathryn Ching, Jeffery McGarvey, David Hodge and Robert Hnasko

Toxins 2013, 5(11), 2212-2226; https://doi.org/10.3390/toxins5112212 - 18 Nov 2013

Cited by 20 | Viewed by 8726

Abstract

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A–H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by [...] Read more.

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A–H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s)for individual antibodies ranging from 10 to 48 × 10⁻¹¹ M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

463 KiB

Open AccessArticle

Faces of a Changing Climate: Semi-Quantitative Multi-Mycotoxin Analysis of Grain Grown in Exceptional Climatic Conditions in Norway

by Silvio Uhlig, Gunnar Sundstøl Eriksen, Ingerd Skow Hofgaard, Rudolf Krska, Eduardo Beltrán and Michael Sulyok

Toxins 2013, 5(10), 1682-1697; https://doi.org/10.3390/toxins5101682 - 27 Sep 2013

Cited by 120 | Viewed by 9579

Abstract

Recent climatological research predicts a significantly wetter climate in Southern Norway as a result of global warming. Thus, the country has already experienced unusually wet summer seasons in the last three years (2010–2012). The aim of this pilot study was to apply an [...] Read more.

Recent climatological research predicts a significantly wetter climate in Southern Norway as a result of global warming. Thus, the country has already experienced unusually wet summer seasons in the last three years (2010–2012). The aim of this pilot study was to apply an existing multi-analyte LC-MS/MS method for the semi-quantitative determination of 320 fungal and bacterial metabolites in Norwegian cereal grain samples from the 2011 growing season. Such knowledge could provide important information for future survey and research programmes in Norway. The method includes all regulated and well-known mycotoxins such as aflatoxins, trichothecenes, ochratoxin A, fumonisins and zearalenone. In addition, a wide range of less studied compounds are included in the method, e.g., Alternaria toxins, ergot alkaloids and other metabolites produced by fungal species within Fusarium, Penicillium and Aspergillus. Altogether, 46 metabolites, all of fungal origin, were detected in the 76 barley, oats and wheat samples. The analyses confirmed the high prevalence and relatively high concentrations of type-A and -B trichothecenes (e.g., deoxynivalenol up to 7230 µg/kg, HT-2 toxin up to 333 µg/kg). Zearalenone was also among the major mycotoxins detected (maximum concentration 1670 µg/kg). Notably, several other Fusarium metabolites such as culmorin, 2-amino-14,16-dimethyloctadecan-3-ol and avenacein Y were co-occurring. Furthermore, the most prevalent Alternaria toxin was alternariol with a maximum concentration of 449 µg/kg. A number of Penicillium and Aspergillus metabolites were also detected in the samples, e.g., sterigmatocystin in concentrations up to 20 µg/kg. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

259 KiB

Open AccessArticle

Non-Linear Relationships between Aflatoxin B₁ Levels and the Biological Response of Monkey Kidney Vero Cells

by Reuven Rasooly, Bradley Hernlem, Xiaohua He and Mendel Friedman

Toxins 2013, 5(8), 1447-1461; https://doi.org/10.3390/toxins5081447 - 14 Aug 2013

Cited by 12 | Viewed by 7410

Abstract

Aflatoxin-producing fungi contaminate food and feed during pre-harvest, storage and processing periods. Once consumed, aflatoxins (AFs) accumulate in tissues, causing illnesses in animals and humans. Most human exposure to AF seems to be a result of consumption of contaminated plant and animal products. [...] Read more.

Aflatoxin-producing fungi contaminate food and feed during pre-harvest, storage and processing periods. Once consumed, aflatoxins (AFs) accumulate in tissues, causing illnesses in animals and humans. Most human exposure to AF seems to be a result of consumption of contaminated plant and animal products. The policy of blending and dilution of grain containing higher levels of aflatoxins with uncontaminated grains for use in animal feed implicitly assumes that the deleterious effects of low levels of the toxins are linearly correlated to concentration. This assumption may not be justified, since it involves extrapolation of these nontoxic levels in feed, which are not of further concern. To develop a better understanding of the significance of low dose effects, in the present study, we developed quantitative methods for the detection of biologically active aflatoxin B₁ (AFB1) in Vero cells by two independent assays: the green fluorescent protein (GFP) assay, as a measure of protein synthesis by the cells, and the microculture tetrazolium (MTT) assay, as a measure of cell viability. The results demonstrate a non-linear dose-response relationship at the cellular level. AFB1 at low concentrations has an opposite biological effect to higher doses that inhibit protein synthesis. Additional studies showed that heat does not affect the stability of AFB1 in milk and that the Vero cell model can be used to determine the presence of bioactive AFB1 in spiked beef, lamb and turkey meat. The implication of the results for the cumulative effects of low amounts of AFB1 in numerous foods is discussed. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

604 KiB

Open AccessArticle

A Label Free Colorimetric Assay for the Detection of Active Botulinum Neurotoxin Type A by SNAP-25 Conjugated Colloidal Gold

by Jennifer Halliwell and Christopher Gwenin

Toxins 2013, 5(8), 1381-1391; https://doi.org/10.3390/toxins5081381 - 6 Aug 2013

Cited by 14 | Viewed by 7925

Abstract

Botulinum neurotoxins are one of the most potent toxins known to man. Current methods of detection involve the quantification of the toxin but do not take into account the percentage of the toxin that is active. At present the assay used for monitoring [...] Read more.

Botulinum neurotoxins are one of the most potent toxins known to man. Current methods of detection involve the quantification of the toxin but do not take into account the percentage of the toxin that is active. At present the assay used for monitoring the activity of the toxin is the mouse bioassay, which is lengthy and has ethical issues due to the use of live animals. This report demonstrates a novel assay that utilises the endopeptidase activity of the toxin to detect Botulinum neurotoxin in a pharmaceutical sample. The cleaving of SNAP-25 is monitored via UV-Visible spectroscopy with a limit of detection of 373 fg/mL and has been further developed into a high throughput method using a microplate reader detecting down to 600 fg/mL of active toxin. The results show clear differences between the toxin product and the placebo, which contains the pharmaceutical excipients human serum albumin and lactose, showing that the assay detects the active form of the toxin. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

311 KiB

Open AccessArticle

Development and Evaluation of Monoclonal Antibodies for the Glucoside of T-2 Toxin (T2-Glc)

by Chris M. Maragos, Cletus Kurtzman, Mark Busman, Neil Price and Susan McCormick

Toxins 2013, 5(7), 1299-1313; https://doi.org/10.3390/toxins5071299 - 19 Jul 2013

Cited by 19 | Viewed by 7858

Abstract

The interactions between fungi and plants can yield metabolites that are toxic in animal systems. Certain fungi are known to produce sesquiterpenoid trichothecenes, such as T-2 toxin, that are biotransformed by several mechanisms including glucosylation. The glucosylated forms have been found in grain [...] Read more.

The interactions between fungi and plants can yield metabolites that are toxic in animal systems. Certain fungi are known to produce sesquiterpenoid trichothecenes, such as T-2 toxin, that are biotransformed by several mechanisms including glucosylation. The glucosylated forms have been found in grain and are of interest as potential reservoirs of T-2 toxin that are not detected by many analytical methods. Hence the glucosides of trichothecenes are often termed “masked” mycotoxins. The glucoside of T-2 toxin (T2-Glc) was linked to keyhole limpet hemocyanin and used to produce antibodies in mice. Ten monoclonal antibody (Mab)-producing hybridoma cell lines were developed. The Mabs were used in immunoassays to detect T2-Glc and T-2 toxin, with midpoints of inhibition curves (IC₅₀s) in the low ng/mL range. Most of the Mabs demonstrated good cross-reactivity to T-2 toxin, with lower recognition of HT-2 toxin. One of the clones (2-13) was further characterized with in-depth cross-reactivity and solvent tolerance studies. Results suggest Mab 2-13 will be useful for the simultaneous detection of T-2 toxin and T2-Glc. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

Review

Jump to: Research

1151 KiB

Open AccessReview

Biotoxin Detection Using Cell-Based Sensors

by Pratik Banerjee, Spyridon Kintzios and Balabhaskar Prabhakarpandian

Toxins 2013, 5(12), 2366-2383; https://doi.org/10.3390/toxins5122366 - 29 Nov 2013

Cited by 48 | Viewed by 14860

Abstract

Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of [...] Read more.

Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of using CBBs for probing biotoxins and toxic agents is that CBBs respond to the toxic exposures in the manner related to actual physiologic responses of the vulnerable subjects. The results obtained from CBBs are based on the toxin-cell interactions, and therefore, reveal functional information (such as mode of action, toxic potency, bioavailability, target tissue or organ, etc.) about the toxin. CBBs incorporate both prokaryotic (bacteria) and eukaryotic (yeast, invertebrate and vertebrate) cells. To create CBB devices, living cells are directly integrated onto the biosensor platform. The sensors report the cellular responses upon exposures to toxins and the resulting cellular signals are transduced by secondary transducers generating optical or electrical signals outputs followed by appropriate read-outs. Examples of the layout and operation of cellular biosensors for detection of selected biotoxins are summarized. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Advances in Toxin Detection

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Related Special Issue

Published Papers (8 papers)

Research

Review

Further Information

Guidelines

MDPI Initiatives

Follow MDPI