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

Poras, Hervé; Ouimet, Tanja; Orng, Sou-Vinh; Fournié‐Zaluski, Marie‐Claude; Popoff, Michel R.; Roques, Bernárd P.

doi:10.1128/aem.00091-09

Cited by 25 publications

(22 citation statements)

References 39 publications

(38 reference statements)

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“…17 Of these, the fluorigenic PL63 is a short, As previously shown using purified BoNT/A, increasing the length of PL63 to reach the α-exosite binding sequence, as in PL50, increases substrate binding. This is clearly evidenced here using LC BoNT/A, revealing a 20-fold increase in the K m value for the longer peptide substrate.…”

Section: Discussionmentioning

confidence: 99%

“…Peptides were synthesized by the solid-phase method, using an ABI 433A automated synthesizer (Applied Biosystems, Foster City, CA), coupled to a 785A programmable absorbance UV detector, based on classical Fmoc chemistry with HBTU/HOBt/DIEA as coupling reagents, as described in Poras et al 17 Purity of the peptides, determined by highperformance liquid chromatography (HPLC), was greater than 98.5%, and their molecular weights (MW) were confirmed by deconvolution of the mass spectra obtained with an electrospray mass spectrometer using Xcalibur software (Thermo Finnigan, San Jose, CA) (see supplemental data for details concerning each peptide): , 5 mM DTT, 1 mg/mL bovine serum albumin [BSA]) with increasing concentrations of either substrate. Reactions with PL63 and PL50 were incubated at 37 °C for either 30 or 10 min, respectively.…”

Section: Peptide Synthesismentioning

confidence: 99%

“…These assays rely on mass spectrometry, 11,12 immunological detection, 13,14 or endopeptidase activity against the entire SNAP-25 or fragments thereof. [15][16][17] The advantage of the latter assay is that it measures and quantifies the active component of the toxin, which is directly responsible for its toxicity. With mounting pressure from the authorities to reduce animal consumption, use of endopeptidase assays in production lines for quality control purposes could prove highly advantageous, insofar as the assay is sensitive enough to detect low doses of toxin.…”

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confidence: 99%

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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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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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Section: Discussionmentioning

confidence: 99%

Section: Peptide Synthesismentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Fluorogenic methods offer a high-throughput alternative to either the mouse bioassay or immuno-MS methods. However, most fluorogenic assays use low-affinity substrates, are more than four orders of magnitude less sensitive than the mouse bioassay, and/or are limited to only a few BoNT serotypes [26][27][28][29][30][31].…”

Section: Introductionmentioning

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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“…Upon substrate cleavage, the two fluorophores are separated, so that the fluorescence of the donor is no longer quenched and can be measured. This principle was used by different groups to detect the activity of different BoNT serotypes with sensitivities between 35 pg/mL and 150 ng/mL depending on serotype, FRET substrate, and assay time used (Anne et al 2001;Dong et al 2004;Rasooly and Do 2008;Pires-Alves et al 2009;Poras et al 2009;Gilmore et al 2011;Ruge et al 2011). The principle has also been implemented into portable devices with sensitivities in the ng/mL range (Sapsford et al 2008;Kostov et al 2009;Sun et al 2010;Balsam et al 2011) and is the basis for commercial substrates like SNAPtide Ò (Shine et al 2002).…”

Section: Endopeptidase Assaysmentioning

confidence: 99%

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

Dorner

Schulz

Kull

et al. 2012

Current Topics in Microbiology and Immunology

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The detection of botulinum neurotoxins (BoNT) is extremely challenging due to their high toxicity and the multiple BoNT variants. To date, seven serotypes with more than 30 subtypes have been described, and even more subtypes are expected to be discovered. The fact that the BoNT molecules are released as large complexes of different size and composition adds further complexity to the issue. Currently, in the diagnostics of botulism, the mouse bioassay (MBA) is still considered as gold standard for the detection of BoNT in complex sample materials. Over the years, different functional, immunological, and spectrometric assays or combinations thereof have been developed, supplemented by DNA-based assays for the detection of the organism. In this review, advantages and limitations of the current technologies will be discussed, highlighting some of the intricacies of real sample analysis.

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

Cited by 25 publications

References 39 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

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

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