Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

Abstract

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...

In Vitro Detection and Quantification of Botulinum Neurotoxin Type E Activity in Avian Blood

“…Ruge et al recently reported the use of fluorogenic substrates, called the BoTest reporters, to develop high-throughput and sensitive assays to detect BoNT endopeptidase activity (37). For the detection of BoNT/A and BoNT/E, the BoTest A/E reporter is composed of SNAP-25 residues 141 to 206 fused to an N-terminal cyan fluorescent protein (CFP) and a C-terminal yellow fluorescent protein (YFP) (13,37).…”

“…Construction of the plasmid encoding the BoTest A/E reporter, pBoTest A/E, is described elsewhere (37). The BoNT/ E-specific reporter plasmid (pBoTest E) was constructed by amplifying pBoTest A/E using phosphorylated primers 5Ј-Pi-CCA ACC AAG AGG CAA CAA AGA TGC-3Ј and 5Ј-Pi-CTT CAT CAA TTC TGG TTT TGT TGG AG-3Ј (where Pi indicates phosphorylated primer), designed to introduce an R198E mutation to deactivate the BoNT/A cleavage site (12).…”

“…All FRET reporter constructs were expressed and purified by double-affinity chromatography as described previously (37). The resulting reporters were quantified by bicinchoninic acid (Pierce, Rockford, IL), divided into single-use aliquots, and stored at Ϫ80°C.…”

“…Other in vitro assays for detection of BoNTs use fluorescent substrates or mass spectrometry coupled with immunological techniques (5,9,13,15,17,20,23,35). Shortcomings of these methods include poor sensitivity, an inability to detect BoNT in complex sample matrices, and expensive equipment requirements (13,37).…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…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).…”