Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B.

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.     

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.     

Black tea extract, thearubigin fraction, counteract the effects of botulinum neurotoxins in mice

Botulinum neurotoxin type A (BoNT/A, 1.5 nM) completely inhibited indirectly evoked twitches in in vitro mouse phrenic nerve-diaphragm preparations within 40 - 45 min. Black tea extract, thearubigin fraction (TRB), mixed with BoNT/A blocked the inhibitory effect of the toxin. The protective effect of TRB extended to botulinum neurotoxins types B and E (BoNT/B and BoNT/E) and tetanus toxin, but not to tetrodotoxin. TRB was also effective against oral toxicity of BoNT/A, B and E. Thus, TRB may be of potential benefit in protecting the paralytic actions of botulinum neurotoxins (BoNTs), but its use is limited by mixing with the toxin.     

Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F.

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.     

Sensing the deadliest toxin: technologies for botulinum neurotoxin detection

Sensitive and rapid detection of botulinum neurotoxins (BoNTs), the most poisonous substances known to date, is essential for studies of medical applications of BoNTs and detection of poisoned food, as well as for response to potential bioterrorist threats. Currently, the most common method of BoNT detection is the mouse bioassay. While this assay is sensitive, it is slow, quite expensive, has limited throughput and requires sacrificing animals. Herein, we discuss and compare recently developed alternative in vitro detection methods and assess their ability to supplement or replace the mouse bioassay in the analysis of complex matrix samples.     

Toxemia in Human Naturally Acquired Botulism

Human botulism is a severe disease characterized by flaccid paralysis and inhibition of certain gland secretions, notably salivary secretions, caused by inhibition of neurotransmitter release. Naturally acquired botulism occurs in three main forms: food-borne botulism by ingestion of preformed botulinum neurotoxin (BoNT) in food, botulism by intestinal colonization (infant botulism and intestinal toxemia botulism in infants above one year and adults), and wound botulism. A rapid laboratory confirmation of botulism is required for the appropriate management of patients. Detection of BoNT in the patient’s sera is the most direct way to address the diagnosis of botulism. Based on previous published reports, botulinum toxemia was identified in about 70% of food-borne and wound botulism cases, and only in about 28% of infant botulism cases, in which the diagnosis is mainly confirmed from stool sample investigation. The presence of BoNT in serum depends on the BoNT amount ingested with contaminated food or produced locally in the intestine or wound, and the timeframe between serum sampling and disease onset. BoNT levels in patient’s sera are most frequently low, requiring a highly sensitive method of detection. Mouse bioassay is still the most used method of botulism identification from serum samples. However, in vitro methods based on BoNT endopeptidase activity with detection by mass spectrometry or immunoassay have been developed and depending on BoNT type, are more sensitive than the mouse bioassay. These new assays show high specificity for individual BoNT types and allow more accurate differentiation between positive toxin sera from botulism and autoimmune neuropathy patients.     

Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN)

The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively.     

A sensitive and useful radioimmunoassay for neurotoxin and its haemagglutinin complex from Clostridium botulinum

A sensitive radioimmunoassay for the detection of botulinum toxin, produced by Clostridium botulinum, was developed. This employs homogeneous botulinum neurotoxin type A and its 125I-labelled derivative of high specific radioactivity, rather than its complex with haemagglutinin as used hitherto. The sensitivity of the assay is 1 ng of neurotoxin per ml, which is equivalent to 80 LD50 units (half-lethal doses) in mice. Neurotoxin and its complex with haemagglutinin were measurable with equal sensitivity when using antibodies against botulinum neurotoxin type A. Specificity of the assay was demonstrated by the lack of response to type B and E botulinum toxins and to heat-inactivated botulinum toxin or extracts of Clostridium sporogenes strain BL46, which contains many surface antigenic determinants common to Clostridium botulinum. Using appropriate conditions, neurotoxin added to fish extract could be quantified accurately, proportionality being observed between the amounts of standard toxin added. In addition, the amounts of toxin species produced by culturing Clostridium botulinum in canned fish was measurable; the values obtained were comparable to those observed by the mouse bioassay. Moreover, the fish samples gave a dose-response curve in the competition radioimmunoassay which was paralleled by the response of botulinum neurotoxin standards. This assay offers the most sensitive, reliable immunological method available for the quantitation of molecular forms of botulinum toxin. As the technique can be used with unpurified fish extracts, it should be widely applicable to different types of samples contaminated with botulinum toxin; furthermore, the clinical diagnosis of human botulism could be substantiated with this method.     

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.     

Primary cell culture for evaluation of botulinum neurotoxin antagonists.

The actions of botulinum neurotoxin (BoNT) were studied on evoked release of the neurotransmitter glycine in primary mouse spinal cord cells. 3[H]-glycine was taken up by cells in physiological solution and released by depolarization with 56 mM K+ in the presence of 2 mM Ca2+. Release of 3[H]-glycine was found to be inhibited by BoNT serotypes A, B and E with similar potency ratios to those observed in the acutely isolated mouse diaphragm muscle. When spinal cord cultures were exposed to BoNT/A for 24 h, inhibition of 3[H]-glycine release was detected at toxin concentrations as low as 10(-14) M, and complete inhibition was observed at concentration >or=10(-12) M. Preincubation of BoNT/A with polyclonal equine antiserum led to antagonism of toxin-induced inhibition of 3[H]-glycine release in spinal cord cells and to protection of mice from the lethal effects of BoNT/A. It is concluded that spinal cord neurons are a useful model for studying botulinum intoxication and for evaluating BoNT antagonists.     

Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection

Human induced pluripotent stem cells (hiPSC) hold great promise for providing various differentiated cell models for in vitro toxigenicity testing. For Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies, several cell models currently exist, but none examine toxin function with species-specific relevance while exhibiting high sensitivity. The most sensitive cell models to date are mouse or rat primary cells and neurons derived from mouse embryonic stem cells, both of which require significant technical expertise for culture preparation. This study describes for the first time the use of hiPSC-derived neurons for BoNT detection. The neurons used in this study were differentiated and cryopreserved by Cellular Dynamics International (Madison, WI) and consist of an almost pure pan-neuronal population of predominantly gamma aminoisobutyric acidergic and glutamatergic neurons. Western blot and quantitative PCR data show that these neurons express all the necessary receptors and substrates for BoNT intoxication. BoNT/A intoxication studies demonstrate that the hiPSC-derived neurons reproducibly and quantitatively detect biologically active BoNT/A with high sensitivity (EC(50) ～0.3 U). Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A complex was demonstrated, and BoNT/A specificity was confirmed through antibody protection studies. A direct comparison of BoNT detection using primary rat spinal cord cells and hiPSC-derived neurons showed equal or increased sensitivity, a steeper dose-response curve and a more complete SNARE protein target cleavage for hiPSC-derived neurons. In summary, these data suggest that neurons derived from hiPSCs provide an ideal and highly sensitive platform for BoNT potency determination, neutralizing antibody detection and for mechanistic studies.     

Isolation and characterization of a neutralizing antibody specific to internalization domain of Clostridium botulinum neurotoxin type B.

Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. In this study, a neutralizing mouse monoclonal antibody against botulinum neurotoxin serotype B (BoNT/B), named BTBH-N1, was developed from mice immunized with BoNT/B toxoid without non-toxic components, which are generally associated with the toxin. Western blot analysis, using recombinant toxin fragments containing light (L), N-terminal half of heavy (HN) and C-terminal half of heavy chains, indicated that BTBH-N1 recognizes linear epitopes located on the HN domain. An in vivo neutralization assay with mice, was conducted to characterize the neutralization capacity of the BTBH-N1. Only 10 microg of BTBH-N1 completely neutralized 20 units (1 unit = one 50% lethal dose) of BoNT/B. Even though the Mab (up to 100 microg) failed to protect mice challenged with 100 units, it significantly prolonged the time to death in a dose dependent manner. BTBH-N1, the first neutralizing antibody against BoNT/B, could be further developed as effective biological therapeutics for preventing and treating botulism, as well as other diseases caused by BoNT/B.     

Measurement of botulinum types A, B and E neurotoxicity using the phrenic nerve-hemidiaphragm: Improved precision with in-bred mice

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. The current method for assessing the potency of botulinum toxin and antitoxins is the mouse LD₅₀ assay. The mouse phrenic nerve-diaphragm assay is an in vitro assay that closely mimics in vivo respiratory paralysis. In this study, we have further improved the assay by using gelatin as a non-frothing alternative to albumin and investigated the effects of botulinum toxin serotypes A, B and E on phrenic nerve-hemidiaphragms from out-bred MF1 and in-bred Balb/c mice. Improved reproducibility was found with in-bred mice. Balb/c mice were also found to be much less sensitive to type B toxin perhaps indicating differences in the expression of receptor components. Hemidiaphragm preparations from Balb/c mice were approximately 7 times more sensitive to type A toxin and 7-12 times more sensitive to type E toxin relative to type B toxin. These findings indicate that when fully optimised the mouse nerve-diaphragm preparation can provide a functional in vitro model for accurate and reproducible assessment of toxin activity.     

An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing the proteolytically generated cleavage product

Tetanus neurotoxin (TeNT¹) is a bacterial protease which specifically cleaves the vesicle protein synaptobrevin-2 (vesicle associated membrane protein-2, VAMP-2). This proteolytic feature of the toxin has been used to develop a sensitive endopeptidase assay for the detection of TeNT activity as an alternative to the in vivo assay for TeNT toxicity. Recombinant synaptobrevin-2 (rSyb2) is immobilized onto a microtiter plate, and the cleavage of immobilized rSyb2 by TeNT is detected with a polyclonal antibody directed against the newly generated C-terminus of the cleavage product. This antibody is shown to be a highly specific tool for detecting rSyb2 proteolysis by TeNT. The method reaches a detection limit of less than 1 pg TeNT/ml. To our knowledge, this is the most sensitive in vitro assay for the detection of TeNT activity, and it is easy to perform. Besides, the assay can also detect the activity of botulinum neurotoxin type B (BoNT/B). The method can be applied to examine the toxicity of TeNT or BoNT/B preparations as well as the influence of chemicals on TeNT and BoNT/B activity. In the future, the assay may also serve as a basis for the replacement of the in vivo safety control of tetanus vaccines.     

Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.     

Mosquito inoculation: an alternative bioassay for toxins

Mosquitoes were evaluated as a bioassay host for several classes of biological toxins. Mosquitoes were sensitive to snake toxic or neurotoxic phospholipase A2 enzymes (but not to nontoxic phospholipase A2 enzymes), cobrotoxin, saxitoxin, microcystin and the scorpion insect sodium channel toxin. Mosquitoes were not sensitive to ricin, diphtheria toxin, anthrax toxin, botulinum toxin, tetanus toxin, conotoxin G or a scorpion sodium channel toxin toxic to mammals. Specific antisera neutralization tests with mosquitoes gave comparable results to those of a mouse assay. The mosquito is a suitable bioassay animal for many, but not all biological toxins, and offers a safer, more efficient and economical assay than mice.     

Comparison of oral toxicological properties of botulinum neurotoxin serotypes A and B

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the foodborne intoxications in humans. BoNTs in nature are associated with non-toxic accessory proteins known as neurotoxin-associated proteins (NAPs), forming large complexes that have been shown to play important roles in oral toxicity. Using mouse intraperitoneal and oral models of botulism, we determined the dose response to both BoNT/B holotoxin and complex toxins, and compared the toxicities of BoNT/B and BoNT/A complexes. Although serotype A and B complexes have similar NAP composition, BoNT/B formed larger-sized complexes, and was approximately 90 times more lethal in mouse oral intoxications than BoNT/A complexes. When normalized by mean lethal dose, mice orally treated with high doses of BoNT/B complex showed a delayed time-to-death when compared with mice treated with BoNT/A complex. Furthermore, we determined the effect of various food matrices on oral toxicity of BoNT/A and BoNT/B complexes. BoNT/B complexes showed lower oral bioavailability in liquid egg matrices when compared to BoNT/A complexes. In summary, our studies revealed several factors that can either enhance or reduce the toxicity and oral bioavailability of BoNTs. Dissecting the complexities of the different BoNT serotypes and their roles in foodborne botulism will lead to a better understanding of toxin biology and aid future food risk assessments.     

The mechanism underlying the protective effect of the thearubigin fraction of black tea (Camellia sinensis) extract against the neuromuscular blocking action of botulinum neurotoxins

The aim of the present study was to elucidate the mechanism of the protective effect of black tea extract, the thearubigin fraction, against the neuromuscular blocking action of botulinum neurotoxin types A, B, and E. The effects of thearubigin fraction extracted from a black tea infusion were examined on the neuromuscular blocking action of botulinum neurotoxin types A, B, and E in mouse phrenic nerve-diaphragm preparations and on the binding of these toxins to rat cerebrocortical synaptosomes. Botulinum neurotoxin type A (1.5 nM), B (6 nM), or E (5 nM) abolished indirect twitches in mouse phrenic nerve-diaphragm preparations within 50, 90, 90 min., respectively. Thearubigin fraction mixed with each toxin blocked the inhibitory effect of the toxins. The specific binding of [125I]botulinum neurotoxin type A, B, or E to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. The elution profile of [125I]botulinum neurotoxin type A, B, or E on Sephadex G-50 column chromatography was different from that of toxin mixed with thearubigin fraction. These findings indicate that thearubigin fraction protects against the neuromuscular blocking action of botulinum neurotoxin types A, B, and E by binding with the toxins.     

A protein chip membrane-capture assay for botulinum neurotoxin activity

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC₅₀s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC₅₀ of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.     

In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics

Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD₅₀ test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1 mouse LD₅₀/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.