Recent Developments in Botulinum Neurotoxins Detection

Abstract

Botulinum neurotoxins (BoNTs) are produced as protein complexes by bacteria of the genus Clostridium that are Gram-positive, anaerobic and spore forming (Clostridium botulinum, C. butyricum, C. baratii and C. argentinense spp.). BoNTs show a high immunological and genetic diversity. Therefore, fast, precise, and more reliable detection methods are still required to monitor outbreaks and ensure surveillance of botulism. The botulinum toxin field also comprises therapeutic uses, basic research studies and biodefense issues. This review presents currently available detection methods, and new methods offering the potential of enhanced precision and reproducibility. While the immunological methods offer a range of benefits, such as rapid analysis time, reproducibility and high sensitivity, their implementation is subject to the availability of suitable tools and reagents, such as specific antibodies. Currently, the mass spectrometry approach is the most sensitive in vitro method for a rapid detection of active or inactive forms of BoNTs. However, these methods require inter-laboratory validation before they can be more widely implemented in reference laboratories. In addition, these surrogate in vitro models also require full validation before they can be used as replacement bioassays of potency. Cell-based assays using neuronal cells in culture recapitulate all functional steps of toxin activity, but are still at various stages of development; they are not yet sufficiently robust, due to high batch-to-batch cell variability. Cell-based assays have a strong potential to replace the mouse bioassay (MBA) in terms of BoNT potency determination in pharmaceutical formulations; they can also help to identify suitable inhibitors while reducing the number of animals used. However, the development of safe countermeasures still requires the use of in vivo studies to complement in vitro immunological or cell-based approaches.

Keywords: botulinum neurotoxins; botulism; cell-based assays; countermeasures; detection; in vitro; in vivo.

Figure 1

Detection principles.

Figure 2

Sequential steps of microfluidic liposome immobilization and toxin detection. Surface modifications allow immobilization of NMN on the surface of the microfluidic channel. Botulinum neurotoxin is drawn through the channel and binds to the receptors on the liposomal membrane. Addition of the low pH buffer to the channel induces conformational changes of the receptor-bound toxin molecules and subsequent HC-mediated translocation of the LC into the liposome lumen. Inside the liposome lumen, LC proteolytically cleaves FRET peptide reporter molecules, which results in unquenching of the FRET pair and allows for fluorescent readout. The resulting increase in fluorescence intensity is directly influenced by the amount of physiologically active toxin molecules present in the original test solution, and therefore provides an estimate of its potency [67].