Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

Dorner, Martin B.; Schulz, Katharina; Kull, Skadi; Dorner, Brigitte G.

doi:10.1007/978-3-662-45790-0_11

Cited by 9 publications

(11 citation statements)

References 261 publications

(156 reference statements)

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“…In efforts to avoid animal suffering, a multitude of BoNT detection methods has been developed with minimal use of animals. These include in vivo simulation assays, such as the hemidiaphragm assay and local injection assays [57,59,62,[78][79][80][81][82], numerous in vitro assays using immunological detection methods, endopeptidase assays, or a combination of the two such as the endopeptidase-mass spectrometry assay [83][84][85][86][87][88], and cell-based assays [89].…”

Section: Considerations For Botulinum Neurotoxin Detection Methodsmentioning

confidence: 99%

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

Pellett

Tepp

Johnson

2019

Toxins

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Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis of botulism, basic research, drug development, potency determination, and detection in clinical, environmental, and food samples. Ideally, a definitive assay for BoNT should reflect the activity of each of the four steps in nerve intoxication. The in vivo mouse bioassay (MBA) is the 'gold standard' for the detection of BoNTs. The MBA is sensitive, robust, semi-quantitative, and reliable within its sensitivity limits. Potential drawbacks with the MBA include assay-to-assay potency variations, especially between laboratories, and false positives or negatives. These limitations can be largely avoided by careful planning and performance. Another detection method that has gained importance in recent years for research and potency determination of pharmaceutical BoNTs is cell-based assays, as these assays can be highly sensitive, quantitative, human-specific, and detect fully functional holotoxins at physiologically relevant concentrations. A myriad of other in vitro BoNT detection methods exist. This review focuses on critical factors and assay limitations of the mouse bioassay and cell-based assays for BoNT detection. Key Contribution:This manuscript reviews two widely used botulinum neurotoxin detection assays, the mouse bioassay and cell-based assays, and the critical factors involved in their methodology as well as interpretation of results. Despite our ability to now reduce animal use and replace the mouse bioassay for certain aspects of botulinum neurotoxin detection, the mouse bioassay is sensitive and is the only in vivo assay considering all aspects of botulinum neurotoxin intoxication on a physiological level as well as pharmacokinetic aspects of the toxins, and thus remains important assay for botulinum neurotoxin detection.Toxins 2019, 11, 713 2 of 23 cells, with preferential entry into motor-neurons. Inside a neuron, the toxins block neurotransmitter release, leading to the long-lasting descending bilateral paralysis characteristic of botulism [6]. Even if applied intramuscularly for therapeutic purposes, a portion of the injected BoNTs, in particular at high doses, can diffuse away from the local injection site leading to distal neuronal effects and at very high doses systemic distribution [7][8][9][10]. BoNTs can also enter human or vertebrate circulation and cause botulism by different routes, including ingestion of contaminated foods, by wound infection with neurotoxigenic clostridia, and by the colonization of the intestinal tract by neurotoxigenic clostridia, causing infant botulism or adult intestinal botulism [11,12]. The latter is rare, as C. botulinum usually does not colonize a healthy intestine with a mature microbiota. The severe symptoms of botulism last from several days to up to a year, depending ...

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Section: Considerations For Botulinum Neurotoxin Detection Methodsmentioning

confidence: 99%

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

Pellett

Tepp

Johnson

2019

Toxins

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“…Furthermore, depending on genomic and strain background as well as environmental conditions such as pH, temperature, redox state etc., the large BoNT family can occur as pure 150 kDa single polypeptide chain or activated di-chain BoNT and as complexes of 300-760 kDa (PTC) in varying NAP-BoNT combinations. This large molecular diversity and BoNTs' extraordinary potency complicates their detection enormously, especially in the presence of complex matrices, such as feces [34].…”

Section: Bonts From Clostridium Botulinum and Other Clostridia Sppmentioning

confidence: 99%

“…An arsenal of different methods has been developed in the BoNT field which can be roughly grouped into (i) immunological assays detecting the presence of the molecule [34], (ii) in vitro biochemical methods such as Endopep-ELISA and Endopep-MS detecting the catalytic activity and the serotype [35,36], and (iii) the functional in vitro cell-based assay [37], ex vivo mice phrenic nerve (MPN) hemidiaphragm assay [38], and in vivo mouse bioassay (MBA) [34] which measure the full biological activity and toxicity. However, apart from the mouse bioassay [39] and one sandwich ELISA [40], none of the other methods had been evaluated in a PT or method performance study until recently.…”

Section: Bonts From Clostridium Botulinum and Other Clostridia Sppmentioning

confidence: 99%

Biological toxins of potential bioterrorism risk: Current status of detection and identification technology

Dorner

Zeleny

Harju

et al. 2016

TrAC Trends in Analytical Chemistry

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“…Während mit Labormethoden beispielsweise Toxine im ein-bis zweistelligen Picogramm-pro-Milliliter-Bereich nachgewiesen werden können [19], sind kommerzielle Lateral Flow Assays (LFA) um einen Faktor 1000 weniger empfindlich und benötigen bestenfalls wenige Nanogramm pro Milliliter für den Toxinnachweis [20]. Der Hauptgrund hierfür liegt im direkten Nachweis der Antikör-perbindung, wohingegen bei Labormethoden enzymatische Signalamplifikationsschritte zu einer deutlichen Steigerung der Sensitivität führen [21]. Zwar existieren auch deutlich empfindlichere Varianten immunologischer Schnelltests wie die Immunfiltration [22], aufgrund des erhöhten Präparationsaufwands wird die Durchführung dieser Tests jedoch häufig von Einsatzkräften als zu komplex empfunden.…”

Section: Immunologische Schnelldetektionsverfahrenunclassified