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...

c Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-l samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices.

Anti-WI-1 antibodies are readily detected by RIA in dogs with blastomycosis. Titers become high, decline during treatment, and persist for months. Anti-A antibodies are sometimes detected with AGID, but these decrease quickly.

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