Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml(-1) was achieved in culture supernatants.     

Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep–MS assay

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD₅₀/ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples.     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

An assay for botulinum toxin types A, B and F that requires both functional binding and catalytic activities within the neurotoxin

Aim: To develop a novel assay technique for the botulinum neurotoxin family (BoNTs) which is dependent on both the endopeptidase and receptor‐binding activities of the BoNTs and which is insensitive to antigenic variation with the toxin family. Methods and Results: An endopeptidase activity, receptor‐binding assay (EARB assay) has been developed which captures biologically active toxin from media using brain synaptosomes. After capture, the bound toxin can be incubated with its substrate, and cleavage detected using serotype‐specific antibodies raised against the cleaved product of each toxin serotype. The EARB assay was assessed using a range of BoNT serotypes and subtypes. For BoNT/A, detection limits for subtypes A₁, A₂ and A₃ were 0·5, 3 and 10 MLD₅₀ ml^?1, respectively. The limit of detection for BoNT/B₁ was 5 MLD₅₀ ml^?1 and a novel antibody‐based endopeptidase assay for BoNT/F detected toxin at 0·5 MLD₅₀ ml^?1. All these BoNTs can be captured from media containing up to 10% serum without loss of sensitivity. BoNT/A₁ could also be detected in dilutions of a lactose‐ containing formulation similar to that used for clinical preparations of the toxin. Different serotypes were found to possess different optimal cleavage pHs (pH 6·5 for A₁, pH 7·4 for B₁). Conclusions: The EARB assay has been shown to be able to detect a broad range of BoNT serotypes and subtypes from various media. Significance and Impact of the Study: The EARB assay system described is the first convenient in vitro assay system described which is requires multiple functional biological activities with the BoNTs. The assay will have applications in instances where it is essential or desirable to distinguish biologically active from inactive neurotoxin.     

Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A–G) gene complex

Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A–G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A–G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n = 27). Using purified DNA, the assay had a sensitivity of 4–95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 10²–10³ CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days).     

Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD₅₀]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD₅₀) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD₅₀), and 117 pg/ml for BoNT/F (less than 1 LD₅₀) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.     

High level expression of the light chain of botulinum neurotoxin serotype C1 and an efficient HPLC assay to monitor its proteolytic activity

Botulinum neurotoxins (serotypes BoNT/A–BoNT/G) induce botulism, a disease leading to flaccid paralysis. These serotypes are highly specific in their proteolytic cleavage of SNAP-25 (synaptosomal-associated protein of 25 kDa), VAMP (vesicle associated membrane protein) or syntaxin. The catalytic domain (light chain, LC) of the neurotoxin has a Zn²⁺ dependent endopeptidase activity. In order to design drugs and inhibitors against these toxins, high level overexpression and characterization of LC of BoNTs along with the development of assays to monitor their proteolytic activity becomes important. Using the auto-induction method, we attained a high level expression of BoNT/C1(1–430) yielding more than 30 mg protein per 500 ml culture. We also developed an efficient assay to measure the activity of serotype C1 based on a HPLC method. SNAP-25 with varying peptide length has been reported in literature as substrates for BoNT/C1 proteolysis signifying the importance of remote exosites in BoNT/C1 required for activity. Here, we show that a 17-mer peptide corresponding to residues 187–203 of SNAP-25, which has earlier been shown to be a substrate for BoNT/A, can be used as a substrate for quantifying the activity of BoNT/C1(1–430). There was no pH dependence for the proteolysis, however the presence of dithiothreitol is essential for the reaction. Although the 17-mer substrate bound 110-fold less tightly to BoNT/C1(1–430) than SNAP-25, the optimal assay conditions facilitated an increase in the catalytic efficiency of the enzyme by about 5-fold.     

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10³ and 10⁵ CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.Botulinum neurotoxins (BoNTs), the causative agents of botulism, are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven serotypes, A to G. While the botulinum neurotoxins BoNT/A, BoNT/B, BoNT/E, and BoNT/F are known to cause botulism in humans, BoNT/C and BoNT/D are frequently associated with botulism in cattle and birds. Despite its toxicity, BoNT/G has not yet been linked to naturally occurring botulism (26).Botulism is a life-threatening illness caused by food contaminated with BoNT (food-borne botulism), by the uptake and growth of C. botulinum in wounds (wound botulism), or by colonization of the intestinal tract (infant botulism) (14). In addition, C. botulinum and the botulinum neurotoxins are regarded as potential biological warfare agents (8).The gold standard for the detection of BoNTs from food or clinical samples is still the mouse lethality assay, which is highly sensitive but rather time-consuming. In addition to various immunological assays for BoNT detection, several conventional and real-time PCR-based assays for the individual detection of bont genes have been reported (2, 9-12, 15, 20, 23, 27-30). A major improvement is the simultaneous detection of more than one serotype, which results in a reduction of effort and in the materials used. In recent years, both conventional and real-time PCR-based multiplex assays have been developed for the simultaneous detection of C. botulinum serotypes (1, 6, 22, 24). To date, however, no internally controlled multiplex real-time PCR assay for the simultaneous detection and differentiation of all four serotypes relevant for humans has been reported.We describe here a highly specific and sensitive multiplex real-time PCR assay based on the 5′-nuclease TaqMan chemistry (17) for the simultaneous detection of the C. botulinum types A, B, E, and F, including an internal amplification control (IAC). Furthermore, we developed six different singleplex assays based on the TaqMan chemistry for the detection of C. botulinum serotypes A to F. Assays were validated on 42 C. botulinum strains, 57 non-C. botulinum strains, on spiked food samples, and on real samples from cases of botulism in Germany.     

A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic α-Exosite Binding Region

Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K_d) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K_d for BoNT/A Lc of 1.47 × 10^− 10 M and an IC₅₀ (50% inhibitory concentration) of 4.7 × 10^− 10 M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 Å resolution. The structure reveals that the Aa1 VHH binds in the α-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc α-exosite as a target for inhibitor development.     

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase–mass spectrometry (Endopep–MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep–MS assay. Our strategy was based on reported BoNT/B–substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.     

Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.
Methods and results:  The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT / A or BoNT / B , were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.
Conclusions:  The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.
Significance and Impact of the Study:  The LAMP assay would be useful for detection of C. botulinum in environmental samples.     

Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain

Background

Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.

Methods and Findings

We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.

Conclusions

An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.     

Rapid product analysis and increased sensitivity for quantitative determinations of botulinum neurotoxin proteolytic activity

The ultimate molecular action of botulinum neurotoxin (BoNT) is a Zn-dependent endoproteolytic activity on one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. There are seven serotypes (A-G) of BoNT having distinct cleavage sites on the SNARE substrates. The proteolytic activity is located on the N-terminal light chain (Lc) domain and is used extensively as the primary target toward therapeutic development against botulism. Here we describe an improved method using ultra-performance liquid chromatography (UPLC) whereby quantitative data were obtained in 1/10th the time using 1/20th the sample and solvent volumes compared with a widely used high-performance liquid chromatography (HPLC) method. We also synthesized a VAMP (vesicle-associated membrane protein)-based peptide containing an intact V1 motif that was efficiently used as a substrate by BoNT/D Lc. Although serotype C1 cleaves the serotype A substrate at a bond separated by only one residue, we were able to distinguish the two reactions by UPLC. The new method can accurately quantify as low as 7 pmol of the peptide substrates for BoNT serotypes A, B, C1, and D. We also report here that the catalytic efficiency of serotype A can be stimulated 35-fold by the addition of Triton X-100 to the reaction mixture. Combining the use of Triton X-100 with the newly introduced UPLC method, we were able to accurately detect very low levels of proteolytic activity in a very short time. Sensitivity of the assay and accuracy and rapidity of product analysis should greatly augment efforts in therapeutic development.     

Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)^?1cm^?1. These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.     

Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

BackgroundBotulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure.ResultsIn this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD₅₀/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD₅₀ BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay.ConclusionThese scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.     

Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle²⁰²]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH₂ acetylated at positions 156 to 203 [Nop¹⁹⁷, Pya²⁰⁰, Nle²⁰²]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 × 10⁶ M⁻¹ s⁻¹ and 8.85 × 10⁶ M⁻¹ s⁻¹, respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.The botulinum neurotoxin (BoNT) family consists of seven antigenically distinct serotypes, BoNT/A to BoNT/G, which act on the peripheral nervous system (19). Of these toxins, serotypes A, B, E, and F cause botulism in humans, a disease characterized by flaccid muscular paralysis. The neurotoxins are produced as single inactive polypeptides of 150 kDa, which are subsequently processed by proteolytic cleavage into biologically active di-chains (19). These forms consist of an approximately 50-kDa light chain (LC) linked by a disulfide bridge to a 100-kDa heavy chain (HC) that contains two domains, designated the binding and translocation domains. The neurotoxins reach their intracellular targets by translocating the LC into the cytosol after endocytosis via interaction of the HC with a high-affinity membrane-bound receptor complex (9, 20). The LC, which possesses a highly specific zinc-endopeptidase activity (29), then blocks the fusion of synaptic vesicles with the presynaptic membrane by selectively cleaving one of the three polypeptides involved in neuroexocytosis. BoNT/A, for instance, cleaves the 206-amino-acid, 25-kDa synaptosome-associated protein (SNAP-25) exclusively between the Q¹⁹⁷ and R¹⁹⁸ residues, thus inhibiting neurotransmitter release at the neuromuscular junction (37, 38).BoNT/A is recognized as the most toxic serotype; its oral 50% lethal dose (LD₅₀) for humans is estimated at 1 μg/kg of body weight (2). Because of this extreme toxicity and prolonged effect, BoNTs are classified by the Centers for Disease Control and Prevention (CDC) as one of the six highest-risk threat agents for bioterrorism in “category A” (27). In spite of this, BoNT/A and -B are widely used as therapeutic agents for the treatment of muscular and nerve disorders, as well as in the treatment of neurological diseases (14, 15, 28). There is also an increasing use of BoNT/A in esthetics for wrinkle reduction (4). Because of their high toxicity, BoNTs are used at very low concentrations, and procedures to be used for their detection and quantification in toxin preparations for medical applications or in the event of malevolent bioterrorist acts have to be highly sensitive, rapid, and easy to use; the use of all lengthy in vivo assays is excluded (2, 11). The advantage of the currently used pharmacotoxicological mouse LD₅₀ (MLD₅₀) assay, considered the gold standard assay, is that it provides the in vivo toxicity of a given botulinum toxin sample, whatever the nature of the infected medium. However, this assay is time-consuming, requires the use of a large number of animals, and has poor repeatability due to many fluctuant parameters involved in this method (22). Several in vitro assays have been reported for the detection of BoNT/A, relying either on mass spectrometry (3, 16), immunological detection (10, 25), or BoNT/A''s endopeptidase activity (12, 30). The advantage of the endopeptidase assay is that it measures and quantifies the “active” part of the toxin, which is directly responsible for neurotransmission inhibition. Various methods have been developed to quantify the BoNT/A proteolytic activity (12, 23, 32-33). Although some of these assays are very sensitive (11), they cannot be used for the field detection of BoNT/A, as they require a multistep procedure, and they are also not easily amenable to quantification of toxin preparations used for medical applications.In this paper, we have designed novel, specific, high-affinity, mimetic peptide substrates for BoNT/A using the internal-collision-induced fluorescence-quenching technique (13). This technique, the use of which has previously been successful in the design of peptide substrates for other Zn-metallopeptidases, e.g., ECE-1 (18) and BoNT/B (1, 26), involves the introduction of a fluorophore/repressor pair, here the highly fluorescent pyrenylalanine (Pya) along with a nitro-phenylalanine (Nop) repressor residue on each side of the cleavage site. Once the better positions of the fluorophore/repressor pair Pya/Nop were determined using a fragment of the SNAP-25 sequence from amino acids 187 to 203 [(187-203) SNAP-25] (30), the kinetic parameters of the peptide substrate were optimized and the stability of the final substrate, acetylated SNAP-25 from positions 156 to 203 [(Ac-156-203) SNAP-25] (Nop¹⁹⁷, Pya²⁰⁰, Nle²⁰²), also called PL50, was finally improved in PL51 by replacing the oxidizable methionine residues within the sequence with norleucines. Thus, the specificity constants (catalytic constant [k_cat]/Michaelis constant [K_m]) of PL50 and of its analogue PL51 were 2.6 × 10⁶ M⁻¹ s⁻¹ and 8.85 × 10⁶ M⁻¹ s⁻¹, respectively. The use of these novel high-affinity substrates provides a simple, one-step, specific, robust, and rapid enzymatic assay, thus fulfilling all the requirements for BoNT/A field detection and for BoNT/A''s quantification in preparations for medical applications.     

A label-free biosensor assay for botulinum neurotoxin B in food and human serum

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5–9.0). Endoproteolytic cleavage of VAMP2 was detected in 10 min with 2 pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3 h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.     

Development of an In Vitro Bioassay for Clostridium botulinum Type B Neurotoxin in Foods That Is More Sensitive than the Mouse Bioassay

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin’s light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml⁻¹ (0.5 mouse 50% lethal dose ml⁻¹) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.     

Presence of antibotulinum neurotoxin antibodies in selected wild canids in Israel

Serum samples from 35 golden jackals (Canis aureus syriacus), eight wolves (Canis lupus), and four red foxes (Vulpes vulpes) from various regions of Israel were collected during the years 2001-04 and tested for antibodies to Clostridium botulinum neurotoxin (BoNT) types C and D. Antibodies against BoNT types C and D were detected in 10 (29%) and in 3 (9%) of 35 golden jackals, respectively, using enzyme-linked immunosorbent assay. This report describes detection of anti BoNT antibodies in wild canids other than coyotes (Canis latrans) for the first time and demonstrates that C. botulinum type C is prevalent in Israel.     

Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA⁺ OrfX⁻) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA⁻ OrfX⁺) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA⁺ OrfX⁻ cluster (69A and 32A) and one strain with the HA⁻ OrfX⁺ cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.