Comparison of in vivo and in vitro mouse bioassays for botulinum toxin antagonists

Sheridan, Robert E.; Deshpande, Shripad B.; Smith, Theresa J.

doi:10.1002/(sici)1099-1263(199912)19:1+<s29::aid-jat611>3.3.co;2-t

Cited by 16 publications

(7 citation statements)

References 15 publications

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“…One day post treatment, BoNT/A, C and E could be detected at levels lower than 1 MLD 50 (with BoNT/A having significant effect on CMAP at levels as low as 0.03 MLD 50 ). The related in vitro mouse/rat hemidiaphragm muscle contraction assay correlates muscle twitch tensions to the toxin concentration [34]. In this in vitro method, hemidiaphragm preparations with attached phrenic nerves are stimulated with a supramaximal pulse and the resulting tension of muscle twitches is recorded.…”

Section: Mouse Lethality Assaymentioning

confidence: 99%

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Čapek

Dickerson

2010

Toxins

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Sensitive and rapid detection of botulinum neurotoxins (BoNTs), the most poisonous substances known to date, is essential for studies of medical applications of BoNTs and detection of poisoned food, as well as for response to potential bioterrorist threats. Currently, the most common method of BoNT detection is the mouse bioassay. While this assay is sensitive, it is slow, quite expensive, has limited throughput and requires sacrificing animals. Herein, we discuss and compare recently developed alternative in vitro detection methods and assess their ability to supplement or replace the mouse bioassay in the analysis of complex matrix samples.

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Section: Mouse Lethality Assaymentioning

confidence: 99%

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Čapek

Dickerson

2010

Toxins

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“…Estimates of statistical significance were based on unpaired two-tailed t test, with a P value of Ͻ0.05 considered significant, as previously reported (13,40,41). Statistical analysis was performed using SigmaPlot 10 (Systat Software, San Jose, CA).…”

Section: Methodsmentioning

confidence: 99%

Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

Roxas-Duncan

Enyedy

Montgomery

et al. 2009

Antimicrob Agents Chemother

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An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitroanilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 M. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.

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“…As the lethal effects of botulinum toxin result from respiratory paralysis, the use of an isolated tissue responsible for respiration seems a logical choice for an alternative. The mouse phrenic nerve hemidiaphragm assay is becoming established as a clear alternative to in vivo assays for research purposes10, 31, 32 and may eventually become accepted for batch release testing. Like in vivo assays, it requires the fully functional toxin and is the most sensitive functional model for detecting neutralising antibodies (<0.5 mU/ml).…”

Section: In Vitro Functional Modelsmentioning

confidence: 99%

Detection of antibodies against botulinum toxins

et al. 2004

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After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only some of these will have the capacity to neutralise the toxin activity. In fact, the ability of toxoid vaccine to induce toxin neutralising antibodies has provided the basis for the use of therapeutic antitoxins and immunoglobulins for the prophylaxis and treatment of diseases caused by bacterial toxins. Increasing indications for the chronic use of botulinum toxin for therapy have inevitably resulted in concern for patients becoming unresponsive because of the presence of circulating toxin-specific antibodies. Highly sensitive and relevant assays to detect only clinically relevant toxin neutralising antibodies are essential. Although immunoassays often provide the sensitivity, their relevance and specificity is often questioned. The mouse protection LD(50) bioassay is considered most relevant but can often only detect 10 mIU/ml of antitoxin. This sensitivity, although sufficient for confirming protective immunity, is inadequate for patients undergoing toxin therapy. An intramuscular paralysis assay improves the sensitivity to ca. 1 mIU/ml, and a mouse ex vivo diaphragm assay, with sensitivity of < 0.5 mIU/ml, is the most sensitive functional assay to date for this purpose. Alternative approaches for the detection of antibodies to botulinum toxin have included in vitro endopeptidase activity neutralisation. Unlike any other functional assay, this approach is not reliant on serotype-specific antibodies for specificity. Most recent promising developments are focused on cellular assays utilising primary rat embryonic cord cells or more conveniently in vitro differentiated established cell lines such as human neuroblastoma cells.

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Comparison of in vivo and in vitro mouse bioassays for botulinum toxin antagonists

Cited by 16 publications

References 15 publications

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium botulinum Neurotoxin Serotype A

Detection of antibodies against botulinum toxins

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