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.

Botulinum neurotoxin: where are we with detection technologies?

Because of its high toxicity, botulinum neurotoxin (BoNT) poses a significant risk to humans and it represents a possible biological warfare agent. Nevertheless, BoNT serotypes A and B are considered an effective treatment for a variety of neurological disorders. The growing applicability of BoNT as a drug, and its potential use as a biological threat agent, highlight the urgent need to develop sensitive detection assays and therapeutic counter measures. In the last decade, significant progress has been made in BoNT detection technologies but none have fully replaced the mouse lethality assay, the current "gold standard". Recently, new advances in robotics and the availability of new reagents have allowed development of methods for rapid toxin analysis. These technologies while promising need further refinement.

Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity.

Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-µl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices.

Conformation-dependent high-affinity monoclonal antibodies to prion proteins.

Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrP(Sc), an aberrantly folded isoform of the normal, cellular prion protein. Detection of PrP(Sc) commonly relies on immunochemical methods, a strategy hampered by the lack of Abs specific for this disease-causing isoform. In this article, we report the generation of eight mAbs against prion protein (PrP) following immunization of Prnp-null mice with rPrP. The eight mAbs exhibited distinct differential binding to cellular prion protein and PrP(Sc) from different species as well as PrP-derived synthetic peptides. Five of the eight mAbs exhibited binding to discontinuous PrP epitopes, all of which were disrupted by the addition of 2-ME or DTT, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP, whereas the F4-31 mAb bound bovine PrP; the K(D) values for mAbs F4-31 and F20-29 were ~500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-ME in ELISA, Western blots, and histoblots. One conformation-dependent mAb F4-31 increased the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture Ab. These new conformation-dependent mAbs were found to be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-ME created unusually high signal-to-noise ratios.

A rapid method to improve protein detection by indirect ELISA.

The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A.

Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.

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.

Antibody protection against botulinum neurotoxin intoxication in mice.

Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics, and other countermeasures. A sensitive immunoassay for detecting BoNT serotype A (BoNT/A), based on monoclonal antibodies (MAbs) F1-2 and F1-40, has been developed and used in complex matrices. The epitope for F1-2 has been mapped to the heavy chain of BoNT/A, and the epitope of F1-40 has been mapped to the light chain. The ability of these MAbs to provide therapeutic protection against BoNT/A intoxication in mouse intravenous and oral intoxication models was tested. High dosages of individual MAbs protected mice well both pre- and postexposure to BoNT/A holotoxin. A combination therapy consisting of antibodies against both the light and heavy chains of the toxin, however, significantly increased protection, even at a lower MAb dosage. An in vitro peptide assay for measuring toxin activity showed that pretreatment of toxin with these MAbs did not block catalytic activity but instead blocked toxin entry into primary and cultured neuronal cells. The timing of antibody rescue in the mouse intoxication models revealed windows of opportunity for antibody therapeutic treatment that correlated well with the biologic half-life of the toxin in the serum. Knowledge of BoNT intoxication and antibody clearance in these mouse models and understanding of the pharmacokinetics of BoNT are invaluable for future development of antibodies and therapeutics against intoxication by BoNT.

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.

Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA.

Botulinum neurotoxins (BoNT), produced by the anaerobic bacterium Clostridium botulinum, cause severe neuroparalytic disease and are considered the most toxic biological agents known. While botulism is rare in the U.S. it often is fatal if not treated quickly, and recovery is long, requiring intensive treatment. BoNT is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disulfide bond. The 'gold standard' for BoNT detection relies on a mouse bioassay. This is a time consuming (up to 4 days) assay and it lacks specificity, however, it gives a sensitivity (mouse LD(50)) of approximately 10 pg mL(-1). Most BoNT immunoassays are much less sensitive. In this study we describe the development of four high-affinity (dissociation constants (Kd's) in the low pM range) monoclonal antibodies (mAbs) that specifically bind BoNT serotype A (BoNT/A). These antibodies, designated F1-2, F1-5, F1-40, and F2-43 are IgG1 subclass mAbs with kappa light chains and they specifically bind BoNT serotype A. Western blot analyses following SDS-PAGE demonstrate that mAbs F1-2 and F1-5 bind the 100 kDa heavy chain subunit and that mAb F1-40 binds the 50 kDa light chain. The fourth antibody demonstrated strong binding to the 150 kDa holotoxin in the ELISA and on Western blots following electrophoresis on native gels. However binding in Western blot studies was not observed for mAb F2-43 following SDS-PAGE. A highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A was developed using mAbs F1-2 and F1-40. Such an assay represents a realistic, high sensitivity alternative to the mouse bioassay.

Effects of purification on the bioavailability of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.

Detection of botulinum neurotoxin-A activity in food by peptide cleavage assay.

The World Health Organization (WHO) and U.S. Centers for Disease Control and Prevention (CDC) have labeled botulinum toxins as a high priority biological agent that may be used in terrorist attacks against food supplies. Due to this threat there is an increased need to develop fast and effective methods to detect active botulinum neurotoxins (BoNTs). This study reports the successful use of an enzymatic assay employing an internally quenched fluorogenic peptide as a fast, simple and inexpensive alternative to the mouse bioassay. In less than 15 min the assay can detect 0.25 nM BoNT-A in liquid food samples. The detection level is far below the adult human lethal oral dose of 70 microg of toxin. Immunomagnetic beads coated with IgG monoclonal antibodies that target the toxin heavy chain can concentrate the toxin without neutralizing its enzymatic activity, overcoming matrix effects caused by endogenous protease inhibitors and peptidases. This fast and effective assay system could be used for large scale screening to detect BoNT-A.

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.

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.

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.

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.