Botulinum neurotoxins: mode of action and detection

Wictome,; Shone,

doi:10.1046/j.1365-2672.1998.0840s187s.x

Cited by 34 publications

(35 citation statements)

References 36 publications

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“…BoNT/A, for instance, specifically cleaves the 206-amino acid SNAP-25 protein exclusively at the Q 197 R 198 scissile bond, thus inhibiting neurotransmitter release at the neuromuscular junction. 8,9 Procedures to be used for BoNT detection and quantification in toxin preparations for medical and aesthetics applications or in the event of malevolent bioterrorist acts have to be highly sensitive as well as selective. The advantage of the currently used pharmacotoxicological mouse LD50 assay, considered the gold standard assay, is to provide the in vivo toxicity of a given botulinum toxin sample whatever the nature of the infected medium.…”

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confidence: 99%

Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity

et al. 2013

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Detection and quantification of low doses of botulinum toxin serotype A (BoNT/A) in medicinal preparations require precise and sensitive methods. With mounting pressure from governmental authorities to replace the mouse LD50 assay, interest in alternative methods such as the endopeptidase assay, quantifying the toxin active moiety, is growing. Using internal collision-induced fluorescence quenching, Pharmaleads produced peptides encompassing the SNAP-25 cleavage site: a 17-mer (PL63) and a 48-mer (PL50) reaching the previously identified α-exosite, with PL50 showing higher apparent affinity for BoNT/A. Peptide mapping experiments revealed that this increased affinity is mainly due to a connecting peptide sequence between the N-terminus of PL63 and the α-exosite, identifying a new cooperative exosite on BoNT/A. Other endopeptidase substrates available, including SNAPTide and BoTest A/E, are both based on fluorescent resonance energy transfer (FRET) technology. To compare these assays, their limits of detection and quantification were determined using light chain or 150-kDa BoNT/A. Detection limits of PL50 and BoTest were over 100 times better than those using SNAPtide in standard conditions. Although the BoTest possessed a detection limit around 0.2 pM for either BoNT/A form, its quantification limit (9.7 pM) using purified BoNT/A was 12 times inferior to PL50, estimated at 0.8 pM, suitable for medicinal preparation quantification.

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Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity

et al. 2013

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“…The BoNTs are proteins of about 150 kDa, consisting of one light chain of 50 kDa and one heavy chain of 100 kDa, linked together by a disulfide bridge (Wictome & Shone, 1998). The toxin is released from the bacteria as a noncovalent complex with other, non-toxic proteins.…”

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Confirmation of botulism in birds and cattle by the mouse bioassay and Endopep-MS

Hedeland

Moura

Båverud

et al. 2011

Journal of Medical Microbiology

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There have been several outbreaks of botulism among poultry and wild birds in Sweden in recent years. The National Veterinary Institute of Sweden (SVA) has identified botulinum neurotoxin (BoNT)/C1 or the mosaic BoNT/C1D using the mouse bioassay. This is believed to be the first report on the application of the Endopep mass spectrometry (Endopep-MS) method to selected clinical animal (serum and liver) samples and a feed sample that had previously given positive test results with the mouse bioassay. In the mouse bioassay eight of the eleven samples were found to be neutralized by both BoNT/C1 and /D antitoxins; the other three were neutralized only by BoNT/C1 antitoxin, but the mice showed a prolonged survival time when the samples had been treated with /D antitoxin. The Endopep-MS analysis, on the other hand, demonstrated only BoNT/C1 activity for all eleven samples. This suggests that at least eight of the samples were of the chimeric toxin type BoNT/C1D, where the enzymically active site is identical to that of BoNT/C1, while other parts of the protein contain sequences of BoNT/D. This is the first step of a cross-validation between the established mouse bioassay and the Endopep-MS of serotypes BoNT/C1 and /C1D. Endopep-MS is concluded to have potential as an attractive alternative to the mouse bioassay.

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“…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 exclusively between the Q 197 and R 198 residues, thus inhibiting neurotransmitter release at the neuromuscular junction (37,38).…”

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

Poras

Ouimet

Orng

et al. 2009

Appl Environ Microbiol

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Botulinum neurotoxins: mode of action and detection

Cited by 34 publications

References 36 publications

Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity

Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity

Confirmation of botulism in birds and cattle by the mouse bioassay and Endopep-MS

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

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