**9. Cell-based assays**

Cell-based assays measure BoNT receptor binding, translocation, and enzymatic activity and can be *in vitro* alternatives to the mouse bioassay. Several neuronal and non-neuronal cell lines have been analyzed for use in neurotoxin assays. These include the following: BE(2)-M17 cells, chick embryo neuronal cells, neuroblastoma cells, and rat spinal cord cells [47–50]. In general, the endpoint of cell-based assays for BoNT/A is the proteolytic cleavage of its intracellular substrate, the vesicle-trafficking SNARE protein called SNAP-25. Recently, Hubbard et al. [51, 52] described the functional analysis of numerous different biological neurotoxins, including BoNTs, in networked cultures of stem cell–derived central nervous system neurons. The investigators demonstrate synaptic activity in cultured neurons of humans and rodents, suggesting that these could serve as comparable methods to animal studies. Hong et al. [53] have also developed a similar assay using a motor neuron-like continuous cell line. Pathe-Neuschäfer-Rube et al. [54] developed a N-terminal tagged luciferase-expressing neuronal cell line. Luciferase is released from these transfected cells during depolarization, which is blocked by botulinum toxin. Cell-based methods may prove to be equally sensitive, or better, than animal studies and may provide a new alternative for *in vivo* experiments. For example, for the first time, the U.S. Food and Drug Administration approved a cell-based assay developed by the biotechnology company Allergan, Inc. (Irvine, CA, USA) for its use as an alternative to the mouse bioassay. However, the details of the Allergan assay have not been published.

## **10. New antibody and biosensor technologies**

Diamant et al. [55] have used an interesting approach for generating antibodies that have higher specificity against serotypes A, B, and E, and possess neutralizing capabilities. Mice were immunized with a "trivalent mixture" of recombinant fragments of neurotoxins A, B, and E. The method generated numerous different monoclonal antibodies against each serotype. Most of the monoclonal antibodies had higher ELISA titers compared to polyclonal antibodies and had specificities with five orders of magnitude greater specificity. These antibodies also protected against neurotoxin dosages of 10–50 LD50. They also observed a neutralizing synergy when serotype-specific monoclonal antibodies were combined into an oligoclonal mixture.

Detection methods can also utilize highly sensitive antibodies to enrich or enhance sample preparation as well as amplify the signal.  The "captured" toxin molecules are then used in an enzyme assay. Using food matrices, the LOD for ALISSA was observed as low as 50 fg/mL. This is far more sensitive than the mouse bioassay, immunoassay, or enzyme assay and suggests that it may be useful for detecting food contamination. Marconi et al. [58, 59] have also described the use of surface plasmon resonance (SPR) to examine synaptic vesicle capture by antibodies against BoNT substrates, such as SNAP25 and VAMP2. SPR could be used with cultured neurons in 96-well plates incubated with either BoNT/A or BoNT/B and may be an alternative to animal studies. Further development of label-free and optical biosensors for detecting botulinum toxin [61, 62] will provide additional technologies with possible impact on food safety.