Development of Cell-Based Assays to Measure Botulinum Neurotoxin Serotype A Activity Using Cleavage-Sensitive Antibodies

Nuss, Jonathan E.; Ruthel, Gordon; Tressler, Lyal E.; Wanner, Laura M.; Torres-Melendez, Edna; Hale, Martha L.; Bavari, Sina

doi:10.1177/1087057109354779

Cited by 27 publications

(47 citation statements)

References 23 publications

(29 reference statements)

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“…Only an assay that considers these four sequential steps can allow an accurate determination of BoNT potency. As supported here, neuronal cell-based (NCB) assays, which meet all of these requirements, are excellent models for determination of BoNT potency (28,29,(47)(48)(49)(50)(51)(52)(53). However, correlation to the MBA still has to be confirmed for most of these assays, and the presented data suggest that this correlation should be determined for each BoNT subtype and formulation.…”

Section: Discussionmentioning

confidence: 93%

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

et al. 2013

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Botulinum neurotoxins (BoNTs) are synthesized by Clostridium botulinum and exist as seven immunologically distinct serotypes designated A through G. For most serotypes, several subtypes have now been described based on nominal differences in the amino acid sequences. BoNT/A1 is the most well-characterized subtype of the BoNT/A serotype, and many of its properties, including its potency, its prevalence as a food poison, and its utility as a pharmaceutical, have been thoroughly studied. In contrast, much remains unknown of the other BoNT/A subtypes. In this study, BoNT/A subtype 1 (BoNT/A1) to BoNT/A5 were characterized utilizing a mouse bioassay, an in vitro cleavage assay, and several neuronal cell-based assays. The data indicate that BoNT/A1 to -5 have distinct in vitro and in vivo toxicological properties and that, unlike those for BoNT/A1, the neuronal and mouse results for BoNT/A2 to -5 do not correlate with their enzymatic activity. These results indicate that BoNT/A1 to -5 have distinct characteristics, which are of importance for a greater understanding of botulism and for pharmaceutical applications.

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Section: Discussionmentioning

confidence: 93%

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

et al. 2013

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“…Cleavage of SNAP-25 has thus been used to evaluate BoNT/A activity in both in vitro and in vivo models [22]. While a number of bioanalytical methods are available to quantify SNAP-25 concentration, including proteomic techniques involving mass spectrometry, immunoassay platforms have become the method of choice due to their versatility in terms of throughput and amenability for both target-based and phenotypic screens [23]. …”

Section: Importance Of Cell-based Assays For Bont Research and Drumentioning

confidence: 99%

“…To this end, Nuss et al created an antigenic peptide that spanned the BoNT/A cleavage site in SNAP-25 (Gln 197-Arg 198) to generate polyclonal antibodies that only recognized full length SNAP-25. Dual channel western blot and direct ELISA using primary chick neurons clearly denoted that a BoNT/A cleavage-sensitive (BACS) antibody was specific for full length SNAP-25 and that BoNT/A addition abrogates this interaction [23]. SNAP-25 proteolysis mediated by BoNT/E, a related serotype that cleaves 17 amino acids upstream from the BoNT/A scissile bond (between Arg 180 and Ile 181), creates a truncated peptide that also has lost the BACS antigen recognition site.…”

Section: 0 Cell-based Immunoassays To Characterize Bont Activitymentioning

confidence: 99%

Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery

Kiris¹,

Kota²,

Burnett³

et al. 2014

Expert Review of Molecular Diagnostics

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Botulinum neurotoxins (BoNTs) are exceptionally potent inhibitors of neurotransmission, causing muscle paralysis and respiratory failure associated with the disease botulism. Currently, no drugs are available to counter intracellular BoNT poisoning. To develop effective medical treatments, cell-based assays provide a valuable system to identify novel inhibitors in a time- and cost-efficient manner. Consequently, cell-based systems including immortalized cells, primary neurons, and stem-cell derived neurons have been established. Stem cell-derived neurons are highly sensitive to BoNT intoxication and represent an ideal model to study the biological effects of BoNTs. Robust immunoassays are used to quantify BoNT activity and play a central role during inhibitor screening. In this review, we examine recent progress in physiologically relevant cell-based assays and high-throughput screening approaches for the identification of both direct and indirect BoNT inhibitors.

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“…Within the past 5-7 years, cell assays have been developed that exceed the sensitivity of the mouse bioassay (Pellett et al 2010(Pellett et al , 2011Whitemarsh et al 2012;McNutt et al 2011;Nuss et al 2010;Kiris et al 2011). Such cell-based assays now offer a sensitive model for the testing of fully functional BoNTs, as is necessary in the potency testing of pharmaceutical and research preparations of BoNTs.…”

Section: Neuronal Cell-based Assaysmentioning

confidence: 99%

Progress in Cell Based Assays for Botulinum Neurotoxin Detection

Pellett¹

2012

Current Topics in Microbiology and Immunology

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Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the causative agent of botulism, and are widely used as valuable pharmaceuticals. The BoNTs are modular proteins consisting of a heavy chain and a light chain linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a multi-step process including specific cell binding, endocytosis, conformational change in the endosome, translocation of the enzymatic light chain into the cells cytosol, and SNARE target cleavage. The quantitative and reliable potency determination of fully functional BoNTs produced as active pharmaceutical ingredient (API) requires an assay that considers all steps in the intoxication pathway. The in vivo mouse bioassay has for years been the 'gold standard' assay used for this purpose, but it requires the use of large numbers of mice and thus causes associated costs and ethical concerns. Cell-based assays are currently the only in vitro alternative that detect fully functional BoNTs in a single assay and have been utilized for years for research purposes. Within the last 5 years, several cell-based BoNT detection assays have been developed that are able to quantitatively determine BoNT potency with similar or greater sensitivity than the mouse bioassay. These assays now offer an alternative method for BoNT potency determination. Such quantitative and reliable BoNT potency determination is a crucial step in basic research, in the development of pharmaceutical BoNTs, and in the quantitative detection of neutralizing antibodies.

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Development of Cell-Based Assays to Measure Botulinum Neurotoxin Serotype A Activity Using Cleavage-Sensitive Antibodies

Cited by 27 publications

References 23 publications

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery

Progress in Cell Based Assays for Botulinum Neurotoxin Detection

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