Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms.

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine.

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of ß-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography-mass spectrometry (LC-MS) methods.

A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins.

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL-1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for ß-amanitin (ß-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.

Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E.

Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A-G). Most foodborne intoxications are caused by serotypes BoNT/A and BoNT/B. BoNT/E outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of BoNT/E3. Monoclonal antibodies (mAbs) were generated against BoNT/E3 by immunizing with recombinant peptide fragments of the light and heavy chains of BoNT/E3. In all, 12 mAbs where characterized for binding to both the recombinant peptides and holotoxin, as well as their performance in Western blots and sandwich ELISAs. The most sensitive sandwich assay, using different mAbs for capture and detection, exhibited a limit of detection of 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. By employing two different mAbs for capture and detection, a more standardized sandwich assay was constructed. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals.

Influence of Food Matrices on the Stability and Bioavailability of Abrin.

Abrin, a highly toxic plant toxin, is a potential bioterror weapon. Work from our laboratory and others have shown that abrin is highly resistant to both thermal and pH inactivation methods. We sought to evaluate the effectiveness of selected food processing thermal inactivation conditions against abrin in economically important food matrices (whole milk, non-fat milk, liquid egg, and ground beef). The effectiveness of toxin inactivation was measured via three different assays: (1) In vitro cell free translation (CFT) assay, (2) Vero cell culture cytotoxicity; and the in vivo mouse intraperitoneal (ip) bioassay. For both whole and non-fat milk, complete inactivation was achieved at temperatures of ≥ 80 °C for 3 min or 134 °C for 60 s, which were higher than the normal vat/batch pasteurization or the high temperature short time pasteurization (HTST). Toxin inactivation in liquid egg required temperatures of ≥ 74 °C for 3 min higher than suggested temperatures for scrambled eggs (22% solids) and plain whole egg. Additionally, the ground beef (80:20%) matrix was found to be inhibitory for full toxin activity in the mouse bioassay while retaining some activity in both the cell free translation assay and Vero cell culture cytotoxicity assay.

A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin.

One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides. The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides.

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies.

Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.

Abrin Toxicity and Bioavailability after Temperature and pH Treatment.

Abrin, one of most potent toxins known to man, is derived from the rosary pea (jequirity pea), Abrus precatorius and is a potential bioterror weapon. The temperature and pH stability of abrin was evaluated with an in vitro cell free translation (CFT) assay, a Vero cell culture cytotoxicity assay, and an in vivo mouse bioassay. pH treatment of abrin had no detrimental effect on its stability and toxicity as seen either in vitro or in vivo. Abrin exposure to increasing temperatures did not completely abrogate protein translation. In both the cell culture cytotoxicity model and the mouse bioassay, abrin's toxic effects were completely abrogated if the toxin was exposed to temperatures of 74 °C or higher. In the cell culture model, 63 °C-treated abrin had a 30% reduction in cytotoxicity which was validated in the in vivo mouse bioassay with all mice dying but with a slight time-to-death delay as compared to the non-treated abrin control. Since temperature inactivation did not affect abrin's ability to inhibit protein synthesis (A-chain), we hypothesize that high temperature treatment affected abrin's ability to bind to cellular receptors (affecting B-chain). Our results confirm the absolute need to validate in vitro cytotoxicity assays with in vivo mouse bioassays.

A Monoclonal-Monoclonal Antibody Based Capture ELISA for Abrin.

Abrin, one of the most highly potent toxins in the world, is derived from the plant, Abrus precatorius. Because of its high toxicity, it poses potential bioterror risks. Therefore, a need exists for new reagents and technologies that would be able to rapidly detect abrin contamination as well as lead to new therapeutics. We report here a group of abrin-specific monoclonal antibodies (mAbs) that recognize abrin A-chain, intact A-B chain toxin, and agglutinin by Western blot. Additionally, these mAbs were evaluated for their ability to serve as capture antibodies for a sandwich (capture) ELISA. All possible capture-detector pairs were evaluated and the best antibody pair identified and optimized for a capture ELISA. The capture ELISA based on this capture-detector mAb pair had a limit of detection (L.O.D) of ≈1 ng/mL measured using three independent experiments. The assay did not reveal any false positives with extracts containing other potential ribosome-inactivating proteins (RIPs). Thus, this new capture ELISA uses mAbs for both capture and detection; has no cross-reactivity against other plant RIPs; and has a sensitivity comparable to other reported capture ELISAs using polyclonal antibodies as either capture or detector.

Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry.

Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibodies (rAb). This determination can be achieved by sequence analysis of immunoglobulin (Ig) transcripts obtained from a monoclonal antibody (MAb) producing hybridoma and subsequent expression of a rAb. However the polyploidy nature of a hybridoma cell often results in the added expression of aberrant immunoglobulin-like transcripts or even production of anomalous antibodies which can confound production of rAb. An incorrect VR sequence will result in a non-functional rAb and de novo assembly of Ig primary structure without a sequence map is challenging. To address these problems, we have developed a methodology which combines: 1) selective PCR amplification of VR from both the heavy and light chain IgG from hybridoma, 2) molecular cloning and DNA sequence analysis and 3) tandem mass spectrometry (MS/MS) on enzyme digests obtained from the purified IgG. Peptide analysis proceeds by evaluating coverage of the predicted primary protein sequence provided by the initial DNA maps for the VR. This methodology serves to both identify and verify the primary structure of the MAb VR for production as rAb.

Probiotic Microorganisms Inhibit Epithelial Cell Internalization of Botulinum Neurotoxin Serotype A.

Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins known to man and are threats to public health and safety. Previous work from our laboratory showed that both BoNT serotype A complex and holotoxin can bind and transit through the intestinal epithelia to disseminate in the blood. The timing of BoNT/A toxin internalization was shown to be comparable in both the Caco-2 in vitro cell culture and in the oral mouse intoxication models. Probiotic microorganisms have been extensively studied for their beneficial effects in not only maintaining the normal gut mucosa but also protection from allergens, pathogens, and toxins. In this study, we evaluate whether probiotic microorganisms will block BoNT/A uptake in the in vitro cell culture system using Caco-2 cells. Several probiotics tested (Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri) blocked BoNT/A uptake in a dose-dependent manner whereas a non-probiotic strain of Escherichia coli did not. We also showed that inhibition of BoNT/A uptake was not due to the degradation of BoNT/A nor by sequestration of toxin via binding to probiotics. These results show for the first time that probiotic treatment can inhibit BoNT/A binding and internalization in vitro and may lead to the development of new therapies.

Rapid Microfluidic Assay for the Detection of Botulinum Neurotoxin in Animal Sera.

Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 µL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL(-1) limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings.

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

Hybridoma Technology.

The generation of hybridoma cell lines by the fusion of splenocytes from immunized mice with immortal myeloma cells is a well-established method for the production of monoclonal antibodies. Although other methods have emerged as an effective alternative for the generation of monoclonal antibodies, the use of hybridoma technology remains a viable technique that is accessible to a wide number of laboratories that perform basic cell biological research. Hybridoma technology represents a relatively simple procedure at minimal cost for the continuous production of native whole immunoglobulins. This chapter will describe the materials and methodologies needed for the successful generation of monoclonal antibody (mAb)-producing hybridoma cell lines against target antigens.

A Double-Sandwich ELISA for Identification of Monoclonal Antibodies Suitable for Sandwich Immunoassays.

The sandwich immunoassay (sELISA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated into the test define most of the performance parameters of any subsequent immunoassay regardless of the assay format: traditional ELISA, lateral-flow immunoassay, various bead-based assays, antibody-based biosensors, or the reporting label. Here we describe an approach for identifying monoclonal antibodies (mAbs) suitable for use as capture antibodies and detector antibodies in a sELISA targeting bacterial protein toxins. The approach was designed for early identification of monoclonal antibodies (mAbs), in the initial hybridoma screen.

Detection of shiga toxins by lateral flow assay.

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia.

Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins. Botulinum neurotoxins associate with neurotoxin-associated proteins (NAPs) forming large complexes that are protected from the harsh environment of the gastrointestinal tract. However, it is still unclear how BoNT complexes as large as 900 kDa traverse the epithelial barrier and what role NAPs play in toxin translocation. In this study, we examined the transit of BoNT serotype A (BoNT/A) holotoxin, complex and recombinantly purified NAP complex through cultured and polarized Caco-2 cells and, for the first time, in the small mouse intestine. Botulinum neurotoxin serotype A and NAPs in the toxin complex were detectable inside intestinal cells beginning at 2 h post intoxication. Appearance of the BoNT/A holotoxin signal was slower, with detection starting at 4-6 h. This indicated that the holotoxin alone was sufficient for entry but the presence of NAPs enhanced the rate of entry. Botulinum neurotoxin serotype A detection peaked at approximately 6 and 8 h for complex and holotoxin, respectively, and thereafter began to disperse with some toxin remaining in the epithelia after 24 h. Purified HA complexes alone were also internalized and followed a similar time course to that of BoNT/A complex internalization. However, recombinant HA complexes did not enhance BoNT/A holotoxin entry in the absence of a physical link with BoNT/A. We propose a model for BoNT/A toxin complex translocation whereby toxin complex entry is facilitated by NAPs in a receptor-mediated mechanism. Understanding the intestinal uptake of BoNT complexes will aid the development of new measures to prevent or treat oral intoxications.

Centrifugal microfluidic platform for ultrasensitive detection of botulinum toxin.

We present an innovative centrifugal microfluidic immunoassay platform (SpinDx) to address the urgent biodefense and public health need for ultrasensitive point-of-care/incident detection of botulinum toxin. The simple, sample-to-answer centrifugal microfluidic immunoassay approach is based on binding of toxins to antibody-laden capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by laser-induced fluorescence. A blind, head-to-head comparison study of SpinDx versus the gold-standard mouse bioassay demonstrates 100-fold improvement in sensitivity (limit of detection = 0.09 pg/mL), while achieving total sample-to-answer time of <30 min with 2-µL required volume of the unprocessed sample. We further demonstrate quantification of botulinum toxin in both exogeneous (human blood and serum spiked with toxins) and endogeneous (serum from mice intoxicated via oral, intranasal, and intravenous routes) samples. SpinDx can analyze, without any sample preparation, multiple sample types including whole blood, serum, and food. It is readily expandable to additional analytes as the assay reagents (i.e., the capture beads and detection antibodies) are disconnected from the disk architecture and the reader, facilitating rapid development of new assays. SpinDx can also serve as a general-purpose immunoassay platform applicable to diagnosis of other conditions and diseases.

New high-affinity monoclonal antibodies against Shiga toxin 1 facilitate the detection of hybrid Stx1/Stx2 in vivo.

BACKGROUND: Shiga toxin-producing E. coli (STEC) are a group of common and potentially deadly intestinal pathogens expressing Shiga toxin (Stx) as a primary virulence factor. Of the two types of Stx, Stx2 is responsible for more severe symptoms during infection, while Stx1 is almost identical to the Shiga toxin from Shigella dysenteriae, a ubiquitous pathogen in developing countries. Although antibodies against Stx1 have been reported, few have reached the affinity needed for assembling highly sensitive immunoassays. Sensitive and affordable immunoassays for Stx1 and Stx2 could help improve detection of STEC in livestock, food, the environment, and in clinical samples resulting in improved food safety and human health. METHOD AND FINDINGS: Three new monoclonal antibodies (mAbs) against the B subunit of Stx1 were generated using recombinant toxoid Stx1E167Q and hybridoma technology. These new mAbs recognize all subtypes of Stx1, but do not cross-react with any subtype of Stx2. In addition, they exhibited the ability to neutralize Stx1 toxicity in Vero cell assays. An optimized sandwich ELISA using of a pair of these mAbs had a limit of detection of 8.7 pg/mL, which is superior to any existing assay of this kind. Using one of these Stx1 mAbs in concert with Stx2 mAbs, the presence of hybrid Stx1/Stx2 toxin in the culture media of STEC strains that express both Stx1 and Stx2 was demonstrated. CONCLUSIONS: These new mAbs provide a mix of availability, utility, versatility, and most importantly, increased sensitivity for detection of Stx1. There are numerous potential applications for these mAbs, including low-cost detection assays and therapeutic use. Analysis of hybrid Stx1/2 could provide new insights on the structure, activity, and cellular targets of Shiga toxins.

Use of benzo analogs to enhance antimycotic activity of kresoxim methyl for control of aflatoxigenic fungal pathogens.

The aim of this study was to examine two benzo analogs, octylgallate (OG) and veratraldehyde (VT), as antifungal agents against strains of Aspergillus parasiticus and A.flavus (toxigenic or atoxigenic). Both toxigenic and atoxigenic strains used were capable of producing kojic acid, another cellular secondary product. A. fumigatus was used as a genetic model for this study. When applied independently, OG exhibits considerably higher antifungal activity compared to VT. The minimum inhibitory concentrations (MICs) of OG were 0.3-0.5 mM, while that of VT were 3.0-5.0 mM in agar plate-bioassays. OG or VT in concert with the fungicide kresoxim methyl (Kre-Me; strobilurin) greatly enhanced sensitivity of Aspergillus strains to Kre-Me. The combination with OG also overcame the tolerance of A. fumigatus mitogen-activated protein kinase (MAPK) mutants to Kre-Me. The degree of compound interaction resulting from chemosensitization of the fungi by OG was determined using checkerboard bioassays, where synergistic activity greatly lowered MICs or minimum fungicidal concentrations. However, the control chemosensitizer benzohydroxamic acid, an alternative oxidase inhibitor conventionally applied in concert with strobilurin, did not achieve synergism. The level of antifungal or chemosensitizing activity was also "compound-strain" specific, indicating differential susceptibility of tested strains to OG or VT, and/or heat stress. Besides targeting the antioxidant system, OG also negatively affected the cell wall-integrity pathway, as determined by the inhibition of Saccharomyces cerevisiae cell wall-integrity MAPK pathway mutants. We concluded that certain benzo analogs effectively inhibit fungal growth. They possess chemosensitizing capability to increase efficacy of Kre-Me and thus, could reduce effective dosages of strobilurins and alleviate negative side effects associated with current antifungal practices. OG also exhibits moderate antiaflatoxigenic activity.