Role for standards in assays of botulinum toxins: international collaborative study of three preparations of botulinum type A toxin

Sesardic, Dorothea; Leung, Tong; Das, Rose Gaines

doi:10.1016/j.biologicals.2003.08.001

Cited by 73 publications

(65 citation statements)

References 19 publications

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“…It is not possible to calculate the specific purity of toxin A in proprietary products from LD 50 values provided by the individual manufacturers, unless these values are normalized or standardized. Dr Frevert's conclusions are consistent with other publications on this subject [3][4][5] that agree the LD 50 values indicated on product labels cannot be standardized due to in-house differences in LD 50 protocols, which result in differences in the potency and hence bias the calculation of specific activity.…”

supporting

confidence: 82%

Is it Possible to Accurately Determine Content of Botulinum Neurotoxin Type A in Drug Products?

Sesardic

2010

Drugs R D

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supporting

confidence: 82%

Is it Possible to Accurately Determine Content of Botulinum Neurotoxin Type A in Drug Products?

Sesardic

2010

Drugs R D

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“…The data show that a BoNT/A preparation's specific activity can be determined with an intraassay coefficient of variation (CV) of 9.0-10.5% and an interassay CV of 9.5%. This is considerably more accurate than the mouse bioassay, where CVs are typically on the order of 20-40% depending on the methods and the number of mice used, with the more accurate bioassay methods requiring more than 100 mice for a single determination [20,41].…”

Section: Real-time Detection Of Substrate Disappearancementioning

confidence: 99%

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

Ruge

Dunning

Piazza

et al. 2011

Analytical Biochemistry

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a b s t r a c tMethods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4 h and BoNT/E in 20 h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra-and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists.Ó 2011 Elsevier Inc. All rights reserved.Botulinum neurotoxins (BoNTs), 1 produced by the bacteria of the genus Clostridium, are the most lethal substances known. The zinc-dependent endopeptidases act by entering neurons and cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and, thus, compromise the protein machinery responsible for neurotransmitter release [1][2][3][4][5][6][7][8]. Failure to promptly treat a victim of BoNT poisoning can result in flaccid paralysis, respiratory failure, or death [9]. The combination of extreme potency and a lack of medical treatments other than antitoxin administration and intensive care has made BoNT a biodefense priority requiring the discovery and development of assays to quantify toxins and to identify antagonists to counteract intoxication [10][11][12][13]. Despite their lethality, BoNTs are widely used for cosmetic and pharmaceutical applications due in part to their exquisite specificity for the neuromuscular junction. BoNTs provide relief of muscle tension and pain by inhibiting neurons that cause excessive muscle contractions. Therapeutic preparations of BoNT/A and B serotypes are Food and Drug Administration (FDA) approved for treating glabellar lines, strabismus, cervical dystonia, blepharospasm, cranial nerve VII disorders, and primary axillary hyperhidrosis. Dozens of ''off-label'' BoNT clinical applications have also been documented [14][15][16][17].The mouse bioassay, or lethality test, has been the standard for testing BoNT-containing samples for the past 30 years [18][19][20]. Government agencies use this method for testing food and serum samples for the presence of BoNT, whereas the pharmaceutical industry uses it for quality control and to quantify ''for human use'' BoNT preparations. The test is carried out by injecting mice intraperitoneally with approximately 0.5 ml of sample per mouse and recording the number of deaths over a 1-to 7-day period. The assay is very sensitive, with a detection limit of 5-10 pg for BoNT/A [21,22]. Results for the mouse bioassay are reported in...

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“…Botulinum neurotoxin type A (BoNT/A) is a good example given the exponential increase in the use of this toxin over the past 2 decades for basic research as well as for clinical purposes. The "gold standard" assay for estimating the potency of BoNT/A is the mouse 50% lethal dose assay that provides the in vivo toxicity of a given BoNT/A sample; however, this bioassay is inherently variable (19,26). Factors that affect the potency of BoNT/A activity include differences in toxin formulations, choice of diluent, choice of mouse strain for the bioassay, and the assay design (19).…”

mentioning

confidence: 99%

“…Factors that affect the potency of BoNT/A activity include differences in toxin formulations, choice of diluent, choice of mouse strain for the bioassay, and the assay design (19). The results of an international collaborative study underscored the importance of using standard reference material in the BoNT/A bioassay to reduce interlaboratory variations in toxin potency data (26). Another example arises from a recognized need for standardization of Bordetella pertussis assays, which prompted the formation of an international conference to discuss the harmonization of immunoassays for pertussis diagnostics and vaccine evaluation (29).…”

mentioning

confidence: 99%

The Case for Developing Consensus Standards for Research in Microbial Pathogenesis: Bacillus anthracis Toxins as an Example

et al. 2009

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Role for standards in assays of botulinum toxins: international collaborative study of three preparations of botulinum type A toxin

Cited by 73 publications

References 19 publications

Is it Possible to Accurately Determine Content of Botulinum Neurotoxin Type A in Drug Products?

Is it Possible to Accurately Determine Content of Botulinum Neurotoxin Type A in Drug Products?

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

The Case for Developing Consensus Standards for Research in Microbial Pathogenesis: Bacillus anthracis Toxins as an Example

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