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.     

A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A.

Our goal was to develop a sensitive method for detecting Clostridium botulinum neurotoxin type A (BoNT/A). We were able to detect BoNT/A in the femtogram (10(-15)g) range using an indirect immuno-polymerase chain reaction (immuno-PCR) assay and an indirect sandwich immuno-PCR assay. For the indirect immuno-PCR assay, enzyme-linked immunosorbent assay (ELISA) plates were coated with BoNT/A that was recognized by anti-BoNT/A monoclonal antibody. For the indirect sandwich immuno-PCR assay, the monoclonal antibody was immobilized on ELISA plates for detecting BoNT/A that was recognized by its polyclonal antibodies. Reporter DNA was prepared by PCR amplification using biotinylated 5'-primers, and it was coupled with biotinylated antibodies through streptavidin. In order to increase sensitivity and reduce background noise, the amounts of reporter DNA (ranging from 50 fg to 50 ng) and streptavidin (ranging from 0.125 ng to 8 ng) were optimized. Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected BoNT/A as low as 50 fg. These results are a 10(5)-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods. The assays we developed are currently the most sensitive methods for detecting BoNT/A.     

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.     

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.     

Amino acid composition of Clostridium botulinum type B neurotoxin

To characterize type B botulinum neurotoxin based on reliable data on the amino acid composition, three batches of the neurotoxin were analyzed. Each batch was isolated from a separate neurotoxin producing bacterial culture (strain Okra). Two batches were purified by the same method and one was purified by a different method. The toxin preparations were comparable in purity (judged by polyacrylamide gel--sodium dodecyl sulfate electrophoresis) and similar in amino acid composition. The best estimate of the number of amino acid residues per toxin molecule (mol. wt 152,000) was: Asp212,Thr54,Ser83,Glu130,Pro46,Gly61++ +,Ala44,Val54,CyS11,Met23,Ile144,Leu107 , Tyr81,Phe77,Lys118,His7,Arg39,Trp18.     

Avian eyelid assay, a new diagnostic method for detecting botulinum neurotoxin serotypes A, B and E.

Based upon botulinum neurotoxins' (BoNT) mechanism of action, a novel, rapid, and sensitive avian eyelid assay was developed to detect Clostridium botulinum neurotoxin serotypes A, B and E in assay buffer and mimic samples. It showed that chick was the most optimal model of 20-selected laboratory, non-laboratory animals. The eyelid closure of chick was the indicator symptom for positive results. The detection limits achieved range from 5 to 250 mouse LD(50) for toxin types A, B, and E in a buffer system and mimic samples. No cross reactivity occurred when using staphylococcal enterotoxin B, diphtheria toxin and nerve agent sarin, but cross reactivity was obtained in more than 6h for using high dose of tetanus toxin. This cross reactivity can be differentiated by BoNT neutralization tests with a serotype-specific antiserum in parallel. The avian eyelid assay can be performed within as short a time as 0.4-6 h. We report here the development of avian eyelid assay is the second animal bioassay for the detection of toxin types A, B, and E which approaches the sensitivity of the mouse bioassay, and is simple to perform as well as rapid to yield results.     

A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice

This study compared the respective intramuscular (IM) safety margins of two preparations of botulinum toxin (BTX) serotype A and one preparation each of BTX serotypes B and F in mice. Mice received an IM injection (0–200 U kg⁻¹ body weight) of BTX-A (BOTOX® or DYSPORT®), an experimental preparation of BTX-B (WAKO Chemicals, Inc.), or an experimental preparation of BTX-F (WAKO). An observer who was masked to treatment scored muscle weakness using the Digit Abduction Scoring (DAS) assay. Peak DAS responses were plotted and IM ED₅₀ values calculated. The safety margin for each BTX preparation was calculated as a ratio of the IM median lethal dose after hind limb injection to the median effective dose in the DAS assay (IM LD₅₀/IM ED₅₀). Experiments were repeated 4–6-times for each preparation (10 mice/dose). Mean safety margin values were highest for BTX-F (WAKO; 16.7±3.9) and one of the BTX-A preparations (BOTOX®; 13.9±1.7). Mean safety margins values for the other BTX-A preparation (DYSPORT®) and BTX-B (WAKO) were significantly lower (7.6±0.9 and 4.8±1.1, respectively). Thus, the BTX preparations exhibited different safety margins in mice. These results support the hypothesis that the preparations are unique therapeutics and are not interchangeable based on a simple dose ratio.     

Development of potent inhibitors of botulinum neurotoxin type B

Botulinum neurotoxins are the most potent toxins known to date. They are zinc-metalloproteases able to cleave selectively an essential component of neurotransmitter exocytosis, causing the syndrome of botulism characterized by a flaccid paralysis. There is a great interest in designing antagonists of the action of these toxins. One way is to inhibit their catalytic activity. In this study, we report the design of such inhibitors directed toward BoNT/B. A study of the S(1) subsite specificity, using several beta-amino thiols, has shown that this subsite prefers a p-carboxybenzyl moiety. The specificity of the S(1)' and S(2)' subsites was studied using two libraries of pseudotripeptides containing the S(1) synthon derived from the best beta-amino thiol tested. Finally, a selection of various non natural amino acids for the recognition of the "prime" domain led to the most potent inhibitor of BoNT/B described to date with a K(i) value of 20 nM.     

Clostridium botulinum type D neurotoxin: purification and detection

A method is reported for the purification of type D botulinum toxin using a combination of low and high pressure ion exchange chromatography. The procedure produced homogeneous toxin in its free form in 3 days, with a specific toxicity in mice of 5.4 x 10(7) LD50/mg protein. Polyclonal antibodies against the pure toxin were raised in rabbits and detected the toxin in both ELISA and western blotting. The antibodies also detected type C1 botulinum toxin using these techniques, confirming the presence of cross-reacting antigenic determinants in these two proteins.     

Amino acid composition of Clostridium botulinum type F neurotoxin

To develop reliable data on the amino acid composition of type F botulinum neurotoxin, three batches of the neurotoxin were analyzed. Each batch was isolated from a separate neurotoxin producing bacterial culture. Two batches had inoculum from one source and the other batch one from a different source. Two batches of the neurotoxin were purified by the same method and one was purified by a different method. The neurotoxin preparations were found comparable in purity and similar in amino acid composition. The best estimate of number of amino acid residues per neurotoxin molecule (mol. wt. 155,000) was: Asp218 Thr80 Ser105 Glu128 Pro47 Gly69 Ala47 Val72 CyS9 Met14 Ile128 Leu104 Tyr86 Phe60 Lys90 His13 Arg51 Trp23.     

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.     

Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle.

Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD(50) units of BoNT/A or 20 mouse LD(50) units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to monitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast, tensions in muscles injected with BoNT/A returned to only 2% and 12% of control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracellular stability of catalytically active BoNT/A light chain.     

Disulfide bond reduction corresponds to dimerization and hydrophobi-city changes of Clostridium botulinum type A neurotoxin

AIM: To determine the structure factors that mediate the intoxication process of botulinum neurotoxin type A (BoNT/A). METHODS: Triton X-114 phase separation experiments and 1-anilino-8-naphthalene sulfonate binding assay were used to study the structural factor that corresponds to the hydrophobicity change of BoNT/A. In addition, sucrose density gradient centrifugation and a chemical crosslinking study were employed to determine the quaternary structure of BoNT/A. RESULTS: Our results demonstrated that in other than acidic conditions, the disulfide reduction is the structural factor that corresponds to the hydrophobicity change of BoNT/A. The quaternary structure of BoNT/A exists as a dimmer in acidic solution (pH 4.5), although the monomeric structure of BoNT/A was reported based on X-ray crystallography. CONCLUSION: Disulfide bond reduction is critical for BoNT/A's channel formation and ability to cross endosome membranes. This result implies that compounds that block this disulfide bond reduction may serve as potential therapeutic agents for botulism.     

Rapid microplate assay for monitoring botulinum neurotoxin B catalytic activity

The binding activity of a rabbit polyclonal antiserum raised against a 51-residue peptide (P51) homologous to human VAMP2 (residues 44-94) was examined. Human VAMP2 is an 18-kDa protein located on the external membrane of small synaptic vesicles and is targeted by four of the seven botulinum neurotoxin (BoNT) serotypes (B, D, F and G). The antiserum, designated anti-P51, recognized P51 but exhibited little cross-reactivity with the two cleavage products that result from BoNT/B-mediated proteolysis of P51. The larger of these fragments, designated as P33 (residues 44-76), exhibited a weak but measurable interaction with the antiserum. The smaller cleavage product, designated as P18 (residues 77-94), was not recognized by the antiserum. Anti-P51 was used to monitor BoNT/B light chain (LC)-mediated cleavage of P51 using an indirect ELISA. The serine protease inhibitor phenylmethylsulfonyl fluoride did not inhibit BoNT/B activity, but the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) and the elastase inhibitor 7- N -phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (ICD 1578) produced complete blockade of BoNT/B LC action. Under ideal conditions, it will be possible to evaluate up to seven candidate anti-BoNT/B drugs in triplicate at four concentrations using a single 96-well microtiter plate. These findings indicate that the ELISA will be suitable for rapid screening of BoNT/B inhibitors.     

Stability of the Clostridium botulinum type A neurotoxin complex: an empirical phase diagram based approach

Clostridium botulinum type A neurotoxin (BoNT/A complex) is of great interest to the pharmaceutical industry. The drug itself is a natural complex of the toxin and a number of associated proteins. Surprisingly, relatively little is known about the exact structure and stability of the 900 kDa BoNT/A complex and its component proteins with the exception of the 150 kDa neurotoxin. In this study we describe the relative stability of the BoNT/A complex, the neurotoxin, and its associated proteins over a wide range of temperature and pH employing circular dichroism, intrinsic and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and static light scattering. The data suggest a strong stabilizing effect of the associated proteins on the neurotoxin component. This data is compiled into empirical phase diagrams which permit the simultaneous visualization of multiple data sets over a wide range of conditions.     

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.     

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.