Rapid and Sensitive Nano-Immunosensors for Botulinum.

Botulinum is a deadly bacterial toxin that causes neuroparalytic disease. However, appropriate tools to detect trace toxic proteins are scarce. This study presents a bead-based diffusometric technique for the rapid, simple, and quantitative detection of biological toxins. Functionalized particles called nano-immunosensors were fabricated by forming sandwiched immunocomplexes comprising Au nanoparticles (AuNPs), toxic proteins, and antibodies on fluorescent probe particles. Particle diffusivity tended to decline with increasing concentration of the target proteins. Calibration curves of purified botulinum toxins (0.01-500 ng/mL) were obtained from whole milk and bovine serum, and results suggested that measurement was independent of the background matrix. The activity of botulinum toxin was evaluated by coating synaptosomal-associated protein 25 (SNAP-25) on fluorescent probe particles. AuNP-conjugated antibodies attached to the probe particles when SNAP-25 proteins were cleaved by active botulinum. Thus, toxicity could be detected from slight changes in diffusivity. A short measurement time of 2 min and a limit of detection of 10 pg/mL were achieved. The nano-immunosensors demonstrated rapid biosensing capability and met the demands of onsite screening for food safety, medical instrument hygiene, and cosmetic surgery products.

Rapid Microfluidic Assay for the Detection of Botulinum Neurotoxin in Animal Sera.

Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 µL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL(-1) limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings.

Early diagnosis and treatment in a child with foodborne botulism.

INTRODUCTION: Foodborne botulism is a neuroparalytic disease caused by ingestion of food contaminated with botulinum toxins. Despite rare the mortality rate is high if untreated. Diagnosis of botulism is still a challenge for clinician, due to the variability of clinical manifestations and disease course. We report on a child with type B botulin intoxication who was early diagnosed and treated underlining that clinical suspicion is crucial to start prompt treatment. CASE PRESENTATION: An 11-year-old boy presented with bilateral ptosis and mydriasis, dry mouth, difficulty in swallowing, dysphonia, urine retention and constipation. Clear sensorium and no fever were observed. Immediately the suspicion of botulism was risen and botulinum antitoxin was administered. 3 days later serum and rectal samples tested positive for Clostridium botulinum. The patient completely recovered when discharged from hospital. DISCUSSION: Foodborne botulism is still possible in developed countries. The confirmation test of botulism requires some days. To avoid long delays between intoxication and diagnosis prompt clinical suspicion is thus crucial. The outcome depends on rapid implementation of appropriate management with intensive respiratory care and antitoxin administration.

Electrochemical aptamer scaffold biosensors for detection of botulism and ricin toxins.

Protein toxins present considerable health risks, but detection often requires laborious analysis. Here, we developed electrochemical aptamer biosensors for ricin and botulinum neurotoxins, which display robust and specific signal at nanomolar concentrations and function in dilute serum. These biosensors may aid future efforts for the rapid diagnosis of toxins.

False-Positive Serum Botulism Bioassay in Miller-Fisher Syndrome.

We describe a patient with acute progressive weakness and areflexia. Both botulism and Miller-Fisher variant of Guillain-Barré syndrome were initial diagnostic considerations, and she was treated with intravenous immunoglobulin and botulinum antitoxin. A mouse bioassay was positive for botulinum toxin A, although her clinical course, electrodiagnostic studies, and cerebrospinal fluid findings supported Miller-Fisher syndrome. This patient's atypical features offer points of discussion regarding the evaluation of patients with acute neuromuscular weakness and emphasize the limitations of the botulism bioassay.

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.

A case of type B botulism in a pregnant bitch.

A two-year-old pregnant Gordon setter presented with acute onset of flaccid tetraparesis and respiratory distress. Neurological examination revealed diffuse lower motor neuron dysfunction. Clostridium botulinum neurotoxin B was isolated from the dog's serum. The dog was hospitalised and received supportive care; respiratory function was monitored but positive-pressure ventilation was not required. Recovery was complete within 1 month and parturition occurred without complication 49 days after admission. The puppies delivered lacked any obvious congenital defects and development during the first few months of life was normal. The source of contamination was suspected to be poorly conserved dry food. To the authors' knowledge, this is the first report of C. botulinum neurotoxin B isolation in a dog and the first report of botulism in a pregnant bitch.

Safety aspects of incobotulinumtoxinA high-dose therapy.

Botulinum toxin (BT) used for dystonia and spasticity is dosed according to the number of target muscles and the severity of their muscle hyperactivities. With this no other drug is used in a broader dose range than BT. The upper end of this range, however, still needs to be explored. We wanted to do this by a prospective non-interventional study comparing a randomly selected group of dystonia and spasticity patients receiving incobotulinumtoxinA (Xeomin(®)) high-dose therapy (HD group, n = 100, single dose ≥400 MU) to a control group receiving incobotulinumtoxinA regular-dose therapy (RD group, n = 30, single dose ≤200 MU). At the measurement point all patients were evaluated for systemic BT toxicity, i.e. systemic motor impairment or systemic autonomic dysfunction. HD group patients (56.1 ± 13.8 years, 46 dystonia, 54 spasticity) were treated with Xeomin(®) 570.1 ± 158.9 (min 400, max 1,200) MU during 10.2 ± 7.0 (min 4, max 37) injection series. In dystonia patients the number of target muscles was 46 and the dose per target muscle 56.4 ± 19.1 MU, in spasticity patients 35 and 114.9 ± 67.1 MU. HD and RD group patients reported 58 occurrences of items on the systemic toxicity questionnaire. Generalised weakness, being bedridden, feeling of residual urine and constipation were caused by the underlying tetra- or paraparesis, blurred vision by presbyopia. Dysphagia and dryness of eye were local BT adverse effects. Neurologic examination, serum chemistry and full blood count did not indicate any systemic adverse effects. Elevated serum levels for creatine kinase/MB, creatine kinase and lactate dehydrogenase were most likely iatrogenic artefacts. None of the patients developed antibody-induced therapy failure. Xeomin(®) can be used safely in doses ≥400 MU and up to 1,200 MU without detectable systemic toxicity. This allows expanding the use of BT therapy to patients with more widespread and more severe muscle hyperactivity conditions. Further studies-carefully designed and rigorously monitored-are necessary to explore the threshold dose for clinically detectable systemic toxicity.

Food-borne botulism in Japan in March 2012.

In March 2012, two patients were transported urgently to the hospital in Tottori Prefecture, Japan, because of symptoms suggestive of botulism. Botulinum neurotoxin type A was detected in the clinical specimens and the food consumed by the two patients (vacuum packed adzuki-batto, a sweet adzuki bean soup containing noodles). We were able to make a prompt diagnosis of food botulism associated with the consumption of adzuki-batto, from which the causative pathogen Clostridium botulinum Ab was cultured.

Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism.

Botulinum neurotoxin A (BoNT/A) has intrinsic endoprotease activity specific for SNAP-25, a key protein for presynaptic neurotransmitter release. The inactivation of SNAP-25 by BoNT/A underlies botulism, a rare but potentially fatal disease. There is a crucial need for a rapid and sensitive in vitro serological test for BoNT/A to replace the current in vivo mouse bioassay. Cleavage of SNAP-25 by BoNT/A generates neo-epitopes which can be detected by binding of a monoclonal antibody (mAb10F12) and thus measured by surface plasmon resonance (SPR). We have explored two SPR assay formats, with either mAb10F12 or His6-SNAP-25 coupled to the biosensor chip. When BoNT/A was incubated with SNAP-25 in solution and the reaction products were captured on a mAb-coated chip, a sensitivity of 5 fM (0.1LD50/ml serum) was obtained. However, this configuration required prior immunoprecipitation of BoNT/A. A sensitivity of 0.5 fM in 10% serum (0.1 LD50/ml serum) was attained when SNAP-25 was coupled directly to the chip, followed by sequential injection of BoNT/A samples and mAb10F12 into the flow system to achieve on-chip cleavage and detection, respectively. This latter format detected BoNT/A endoprotease activity in 50-100 µl serum samples from all patients (11/11) with type A botulism within 5h. No false positives occurred in sera from healthy subjects or patients with other neurological diseases. The automated chip-based procedure has excellent specificity and sensitivity, with significant advantages over the mouse bioassay in terms of rapidity, required sample volume and animal ethics.

Persistent neurogenic bladder dysfunction due to infantile botulism.

We present a child, 5 months of age, diagnosed with infantile botulism, showing the signs of neurogenic bladder dysfunction. The patient presented with progressive muscle weakness, hypotonia, suckling and swallowing problems and absent peripheral reflexes at clinical examination. Botulinum neurotoxin type A was detected in her serum, confirming the diagnosis. Starting at day 6, the girl presented with a urinary retention initially necessitating free bladder drainage and subsequently intermittent catheterisation. After 6 weeks in intensive care, the patient recovered but the bladder underactivity persisted. Four months following recovery, a urodynamic evaluation was performed, showing a near normal detrusor activity and normal bladder emptying, and the catheterisation was ceased. At 6 months, the girl was diagnosed with a urinary tract infection and bladder emptying problems, which persisted, and clean intermittent catheterisation was started. The final urodynamic evaluation, a year and a half after her initial presentation, revealed a normal detrusor activity and an adequate bladder emptying.

Identification of botulinum toxin type in clinical samples and foods in Iran.

BACKGROUND: Botulism is a serious neuroparalytic disease caused by toxins of Clostridium botulinum. Botulinum toxin is produced under anaerobic conditions and is one of the most dangerous toxin in the world. Rapid diagnosis of botulism is very essential for successful therapy. In this study, we reviewed data of cases of botulism in Iran from April 2004 through March 2010. MATHERIALS AND METHODS: From a total of 1140 samples of suspected botulism samples, 477 serum, 294 stool, 111 gastric secretions, and 258 food samples were collected from 21 provinces. These samples belonged to 432 distinct patients. All samples were tested for botulism by mouse bioassay, a gold standard method for detection of botulism. RESULTS: From 1140 received samples, 64 (5.6 %) positive samples of botulism were identified. Of these, 14 (21.8 %) cases had toxin type A, seven (11 %) cases had toxin type B, 22 (34.3 %) cases had toxin type E, and seven (11 %) cases had toxin type AB. The toxin type could not been identified in 14 (21.8 %) cases. The highest positive results were in Gilan, Tehran, Golestan, and Hamedan provinces. Seafoods and locally- made cheese were the most implicated foods in type E and type A botulism, respectively. CONCLUSION: Accurate and rapid diagnosis of botulism is very important because every case of botulism can be a public health emergency. During the study period, the median number of positive cases per year was 2.7 (range: one to18). Therefore, it is suggested that all clinicians are required to submit the collected samples from patients with botulism symptoms to the botulism reference laboratory for specific diagnosis and confirmation of botulism.

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin.

A surface plasmon resonance based RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin is reported. Using detoxified recombinant type A botulinum neurotoxin as the surrogate, the aptasensor detects active toxin within 90 min. The detection limit of the aptasensor in phosphate buffered saline, carrot juice, and fat free milk is 5.8 ng/ml, 20.3 ng/ml and 23.4 ng/ml, respectively, while that in 5-fold diluted human serum is 22.5 ng/ml. Recovery of toxin from disparate sample matrices are within 91-116%. Most significant is the ability of this aptasensor to effectively differentiate the natively folded toxin from denatured, inactive toxin, which is important for homeland security surveillance and threat assessment. The aptasensor is stable for more than 30 days and over 400 injections/regeneration cycles. Such an aptasensor holds great promise for rapid detection of active botulinum neurotoxin for field surveillance due to its robustness, stability and reusability.

Use of a new functional dual coating (FDC) assay to measure low toxin levels in serum and food samples following an outbreak of human botulism.

Clostridium botulinum type A toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. The active dichain neurotoxin molecule is composed of a heavy chain (H-chain) of ~100 kDa with the carboxy-terminal end consisting of a receptor-binding (HC) domain, while the amino-terminal (HN) domain is linked by a critical disulfide bond to a light chain (L-chain) of ~50 kDa. Although the mouse bioassay (MBA) is traditionally used to confirm the presence of toxin in serum or food, its sensitivity is insufficient to detect low toxin levels in approximately 30 to 60 % of botulism patients. A novel FDC (functional dual coating) microtitre plate immuno-biochemical assay, which quantifies botulinum toxicity by measuring the HC domain linked with L-chain endopeptidase activity, was modified to allow human serum (lysed or unlysed) to be tested without interference from the matrix, with toxin detection down to 0.03 mouse LD50 per ml serum or 0.13 pg ml(-1) using just 100 µl of clinical samples. The assay was specific for type A toxin and could additionally be applied to whole blood and food samples. Low levels of 1 to 2 mouse LD50 per ml serum of type A toxin were quantified for the first time using the modified FDC assay in two severely intoxicated UK patients who required mechanical ventilation and antitoxin. Toxin levels in recovered food sample extracts were also detected and one MBA-negative sample was found to contain 0.32 LD50 per ml extract. The FDC assay provides a real alternative for public health laboratories to unambiguously confirm all cases of type A botulism and, due to its sensitivity, a promising new tool in toxin pharmacokinetic studies.

Toxin detection in patients' sera by mass spectrometry during two outbreaks of type A Botulism in France.

In two outbreaks of food-borne botulism in France, Clostridium botulinum type A was isolated and characterized from incriminated foods. Botulinum neurotoxin type A was detected in the patients' sera by mouse bioassay and in vitro endopeptidase assay with an immunocapture step and identification of the cleavage products by mass spectrometry.

Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs.

Detection of biologically active botulinum neurotoxin--A in serum using high-throughput FRET-assay.

INTRODUCTION: The goals of this project were to compare fluorescent resonance energy transfer (FRET) assays using a customized FRET substrate (substrate-substrate-A, SSA) with a commercially available FRET substrate (SNAPtide); optimize the assay conditions for SSA for lowest level of detection; and apply SSA to detect botulinum neurotoxin-A (BoNTA) in serum samples. METHODS: Biological activity of BoNTA and light-chain-A (LCA) was verified by murine phrenic nerve-hemidiaphragm bioassay and western blot before use in both FRET assays. The reaction conditions were optimized to determine the smallest amount of toxin that could be detected. A range of serum samples was investigated for interference in the SSA-based FRET assay. Detection of BoNTA from rat serum samples was performed over time. RESULTS: We found that BoNTA and LCA were able to cleave the substrates whereas mutated LCA and a different serotype of BoNT, BoNTB, could not. SSA had significantly more arbitrary fluorescing units compared to the FRET substrate SNAPTide, and the SSA assay could detect 0.1nM of BoNTA or LCA comfortably (p=<0.05) in a 20-µl reaction. No significant interference was observed when serum was present in the reaction buffer. Due to negligible background noise, the SSA FRET assay could detect BoNTA from spiked rat serum even after 256min. DISCUSSION: The greatest advantage of the FRET assay is its extreme rapidity, its cost effectiveness, and unlike ELISA, its ability to detect biologically active toxin. SSA is a better FRET substrate for detecting BoNTA toxin (detected 0.1nM concentration). Because serum present in the assay reaction did not cause any appreciable interference, the assay can be used to detect BoNTA in serum samples. Therefore, the SSA FRET assay can be used for pharmacokinetic and pharmacodynamic studies, screening inhibitors, and detecting BoNTA in serum samples.

Quantification of botulinum neurotoxin serotypes A and B from serum using mass spectrometry.

Botulinum neurotoxins (BoNT) are the deadliest agents known. Previously, we reported an endopeptidase activity based method (Endopep-MS) that detects and differentiates BoNT serotypes A-G. This method uses serotype specific monoclonal antibodies and the specific enzymatic activity of BoNT against peptide substrates which mimic the toxin's natural target. Cleavage products from the reaction are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We have now developed a multiple reaction monitoring method to quantify the biological activity of BoNT serotypes A (BoNT/A) and B (BoNT/B) present in 0.5 mL of serum using electrospray mass spectrometry. The limit of quantification for each serotype is 1 mouse intraperitoneal lethal dose (MIPLD(50)) corresponding to 31 pg of BoNT/A and 15 pg of BoNT/B in this study. This method was applied to serum from rhesus macaques with inhalational botulism following exposure to BoNT/B, showing a maximum activity of 6.0 MIPLD(50)/mL in surviving animals and 653.6 MIPLD(50)/mL in animals that died in the study. The method detects BoNT/B in serum 2-5 h after exposure and up to 14 days. This is the first report of a quantitative method with sufficient sensitivity, selectivity, and low sample size requirements to measure circulating BoNT activity at multiple times during the course of botulism.

[Food-borne botulism].

Botulism is a neuroparalytic disease caused by neurotoxins produced by Clostridium botulinum. Food-borne botulism is a kind of exotoxin-caused food intoxication. Although this disease is rarely reported in Japan now, it is a cause of great concern because of its high mortality rate, and botulism cases should be treated as a public health emergency. Botulism classically presents as acute symmetrical descending flaccid paralysis. Its diagnosis is based on the detection of botulinum toxins in the patient's serum or stool specimens. Electrophysiologic tests of such patients show reduced compound muscle action potentials (CMAPs), low amplitudes and short durations of motor unit potentials (MUPs), and mild waning in repetitive low-frequency stimulations. Single fiber electromyography (EMG) is particularly useful for the diagnosis of botulism. We report a case of food-borne botulism that we had encountered. An 83-year-old man with rapidly progressive diplopia, dysphagia, and tetraplegia was hospitalized; he required intensive care, including artificial ventilatory support. Electrophysiologic tests yielded findings compatible with botulism. We made a clinical diagnosis of food-borne botulism and administered antitoxin on the seventh disease day. The patient's motor symptoms started ameliorating several days after the antitoxin injection. Subsequently, botulinum toxin type A was detected in the patient's serum specimen by using a bioassay, and the type A gene and silent B gene were detected in his serum specimen by using polymerase chain reaction (PCR). C. botulinum was also obtained from stool culture on the 17th and 50th disease days. Botulism is a curable disease if treated early. Although it is a rare condition, it should always be considered in the differential diagnosis of patients with rapid onset of cranial nerve and limb muscle palsies.

Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.