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.     

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.     

Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (K_D) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean K_D 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.     

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 Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model

Botulinum neurotoxins (BoNT) are extremely potent and can induce respiratory failure, requiring long-term intensive care to prevent death. Recombinant monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics. In contrast, equine antitoxin cannot be used prophylactically and has a short half-life. Two three-mAb combinations are in development that specifically neutralize BoNT serotype A (BoNT/A) and B (BoNT/B). The three-mAb combinations addressing a single serotype provided pre-exposure prophylaxis in the guinea pig inhalation model. A lyophilized co-formulation of six mAbs, designated G03-52-01, that addresses both A and B serotypes is in development. Here, we investigated the efficacy of G03-52-01 to protect guinea pigs against an aerosol exposure challenge of BoNT/A1 or BoNT/B1. Previously, it was found that each antibody demonstrated a dose-dependent exposure and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intravenous (IV) injection. Here we show that G03-52-01, in a single IM injection of G03-52-01 administered 48 h pre-exposure, protected guinea pigs against an aerosol challenge of up to 238 LD₅₀s of BoNT/A1 and 191 LD₅₀s of BoNT/B1. These data suggest that a single IM administration of G03-52-01 provides pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A1 or BoNT/B1.     

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.     

Ouabain exacerbates botulinum neurotoxin-induced muscle paralysis via progression of muscle atrophy in mice

Botulinum neurotoxin serotype A (BoNT/A) inhibits acetylcholine release at the neuromuscular junction in isolated muscles, and ouabain can partially block its effect. However, it is not clear whether ouabain attenuates BoNT/A-induced neuromuscular paralysis in vivo. In this work, we investigated the effects of ouabain on BoNT/A-induced neuromuscular paralysis in mice. Ouabain was administered to mice intraperitoneally immediately after a single injection of BoNT/A into skeletal muscle. The effects of ouabain on BoNT/A-induced muscle paralysis were assessed by quantitative monitoring of muscle tension and digit abduction via the digit abduction scoring (DAS) assay. A single administration of ouabain significantly prolonged BoNT/A-induced neuromuscular paralysis. Moreover, consecutive daily injection of ouabain exacerbated BoNT/A-induced neuromuscular paralysis, and led to a significant decrease in both twitch and tetanic forces as assayed in isolated BoNT/A-injected muscles. We next looked at the effects of ouabain on BoNT/A-induced muscle atrophy. Administration of ouabain led to a decrease in the myofibrillar cross-sectional area (CSAs) by 14 post-BoNT/A injection. In addition, repeated administration of ouabain increased mRNA expression levels of ubiquitin ligases, which are markers of muscle atrophy, in BoNT/A-injected muscle. These results suggest that ouabain exacerbates BoNT/A-induced neuromuscular paralysis via a marked progression of BoNT/A-induced muscle atrophy.     

Neutralizing Concentrations of Anti-Botulinum Toxin Antibodies Positively Correlate with Mouse Neutralization Assay Results in a Guinea Pig Model

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency and safety. A three-mAb combination has been developed that specifically neutralizes BoNT serotype A (BoNT/A), and a separate three mAb combination has been developed that specifically neutralizes BoNT serotype B (BoNT/B). A six mAb cocktail, designated G03-52-01, has been developed that combines the anti-BoNT/A and anti-BoNT/B mAbs. The pharmacokinetics and neutralizing antibody concentration (NAC) of G03-52-01 has been determined in guinea pigs, and these parameters were correlated with protection against an inhalation challenge of BoNT/A1 or BoNT/B1. Previously, it was shown that each antibody demonstrated a dose-dependent mAb serum concentration and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intraperitoneal (IP) injection and that a single IM injection of G03-52-01 administered 48 h pre-exposure protected guinea pigs against an inhalation challenge of up to 93 LD₅₀s of BoNT/A1 and 116 LD₅₀s of BoNT/B1. The data presented here advance our understanding of the relationship of the neutralizing NAC to the measured circulating antibody concentration and provide additional support that a single IM or intravenous (IV) administration of G03-52-01 will provide pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A and BoNT/B.     

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.     

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.     

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.     

Correlation of cleavage of SNAP-25 with muscle function in a rat model of Botulinum neurotoxin type A induced paralysis.

Injection of botulinum neurotoxin serotype A (BoNT/A) into muscle results in cleavage of the synaptosomal associated protein of 25 kDa (SNAP-25) and relatively long-term paralysis. However, nerve-terminal sprouting, which appears to require intact SNAP-25, has been reported to occur much earlier. The difference between the long-term paralysis induced by injection of BoNT/A and the short time needed for sprouting led us to investigate the relationship between BoNT/A catalyzed cleavage of SNAP-25 and muscle function. The effect of BoNT/A on SNAP-25 present in nerve endings innervating gastrocnemius muscles of rats was monitored over time. Cleaved SNAP-25 was found in nerve terminals innervating the muscles within 24h of inoculation with BoNT/A and was present more than 2 months later. Comparison of the ratios of cleaved to intact SNAP-25 from the onset of BoNT/A-induced paralysis until function was regained indicated that paralysis was probable when the ratio of cleaved to intact SNAP-25 was greater than 0.35.     

Determination of breviflavone A and B in Epimedium herbs with liquid chromatography–tandem mass spectrometry

Two new types of minor flavonoids, breviflavone A and B, have been recently isolated and identified from Epimedium brevicornu in our previous research. Breviflavone B is a novel flavonoid with potent and specific estrogen receptor (ER) bioactivity. Its positional isomer, breviflavone A, is not ER active. Therefore, it is important to determine the two minor components, breviflavone A and B, in Epimedium herbs. In this report, a robust method for measurement of the two breviflavones in Epimedium ethanolic extracts has been developed by using liquid chromatography tandem mass spectrometry via selected-reaction monitoring (m/z 437 → m/z 367 for breviflavone A and m/z 437 → m/z 351 for breviflavone B) under negative electrospray ionization mode. This method has been successfully used to determine the two breviflavones in ethanolic herbal extracts of five major Epimedium species (E. brevicornu, E. koreanum, E. pubescens, E. sagittatum, and E. wushanese) from various sources. The contents of the two breviflavones range from 0.0181 to 0.1791% for breviflavone A and 0.0026 to 0.0252% for breviflavone B in the dried ethanolic extracts of those Epimedium herbal samples.     

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.     

Recommended Mass Spectrometry-Based Strategies to Identify Botulinum Neurotoxin-Containing Samples

Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between 0.1 μg to 70 μg, so it is important to enable detection of small amounts of these toxins. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric‑based endopeptidase method to detect, differentiate, and quantify BoNT immunoaffinity purified from complex matrices. In this work, we describe the application of Endopep-MS for the analysis of thirteen blinded samples supplied as part of the EQuATox proficiency test. This method successfully identified the presence or absence of BoNT in all thirteen samples and was able to successfully differentiate the serotype of BoNT present in the samples, which included matrices such as buffer, milk, meat extract, and serum. Furthermore, the method yielded quantitative results which had z-scores in the range of −3 to +3 for quantification of BoNT/A containing samples. These results indicate that Endopep-MS is an excellent technique for detection, differentiation, and quantification of BoNT in complex matrices.     

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.     

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.     

Botulinum Neurotoxin Chimeras Suppress Stimulation by Capsaicin of Rat Trigeminal Sensory Neurons In Vivo and In Vitro

Chimeras of botulinum neurotoxin (BoNT) serotype A (/A) combined with /E protease might possess improved analgesic properties relative to either parent, due to inheriting the sensory neurotropism of the former with more extensive disabling of SNAP-25 from the latter. Hence, fusions of /E protease light chain (LC) to whole BoNT/A (LC/E-BoNT/A), and of the LC plus translocation domain (H_N) of /E with the neuronal acceptor binding moiety (H_C) of /A (BoNT/EA), created previously by gene recombination and expression in E. coli., were used. LC/E-BoNT/A (75 units/kg) injected into the whisker pad of rats seemed devoid of systemic toxicity, as reflected by an absence of weight loss, but inhibited the nocifensive behavior (grooming, freezing, and reduced mobility) induced by activating TRPV1 with capsaicin, injected at various days thereafter. No sex-related differences were observed. c-Fos expression was increased five-fold in the trigeminal nucleus caudalis ipsi-lateral to capsaicin injection, relative to the contra-lateral side and vehicle-treated controls, and this increase was virtually prevented by LC/E-BoNT/A. In vitro, LC/E-BoNT/A or /EA diminished CGRP exocytosis from rat neonate trigeminal ganglionic neurons stimulated with up to 1 µM capsaicin, whereas BoNT/A only substantially reduced the release in response to 0.1 µM or less of the stimulant, in accordance with the /E protease being known to prevent fusion of exocytotic vesicles.     

Sheep Monoclonal Antibodies Prevent Systemic Effects of Botulinum Neurotoxin A1

Botulinum neurotoxin (BoNT) is responsible for causing botulism, a potentially fatal disease characterized by paralysis of skeletal muscle. Existing specific treatments include polyclonal antisera derived from immunized humans or horses. Both preparations have similar drawbacks, including limited supply, risk of adverse effects and batch to batch variation. Here, we describe a panel of six highly protective sheep monoclonal antibodies (SMAbs) derived from sheep immunized with BoNT/A1 toxoid (SMAbs 2G11, 4F7) or BoNT/A1 heavy chain C-terminus (HcC) (SMAbs 1G4, 5E2, 5F7, 16F9) with or without subsequent challenge immunization with BoNT/A1 toxin. Although each SMAb bound BoNT/A1 toxin, differences in specificity for native and recombinant constituents of BoNT/A1 were observed. Structural differences were suggested by pI (5E2 = 8.2; 2G11 = 7.1; 4F7 = 8.8; 1G4 = 7.4; 5F7 = 8.0; 16F9 = 5.1). SMAb protective efficacy vs. 10,000 LD50 BoNT/A1 was evaluated using the mouse lethality assay. Although not protective alone, divalent and trivalent combinations of SMabs, IG4, 5F7 and/or 16F9 were highly protective. Divalent combinations containing 0.5–4 μg/SMAb (1–8 μg total SMAb) were 100% protective against death with only mild signs of botulism observed; relative efficacy of each combination was 1G4 + 5F7 > 1G4 + 16F9 >> 5F7 + 16F9. The trivalent combination of 1G4 + 5F7 + 16F9 at 0.25 μg/SMAb (0.75 μg total SMAb) was 100% protective against clinical signs and death. These results reflect levels of protective potency not reported previously.     

A comparative study for PSP toxins quantification by using MBA and HPLC official methods in shellfish

Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated.A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked.Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction and SPE clean-ups steps may also contribute. By contrast, 9 samples presented a much higher total toxicity by HPLC-FLD than by MBA. These higher results obtained by HPLC-FLD could not only be due to the use of the highest toxicity equivalency factor (TEF) for isomers oxidated into products that coelute when total toxicity of these samples were calculated. Further analyses of results obtained by HPLC-FLD and by MBA with both extracts were done separately.