Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity.

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-µl 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.

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters.

Methods 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 4h and BoNT/E in 20h, 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.