The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100？pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it. 1. Introduction Botulinum neurotoxins (BoNTs) are the most toxic substance known [1]. They are primarily produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, by some strains of Clostridium butyricum and Clostridium baratii [2, 3]. Intoxication with one of the seven distinct serotypes of BoNT (A–G) causes botulism. One of 4 serotypes of BoNT (A, B, E, and F) is usually the cause of human botulism. Exposure to type A neurotoxin (BoNT/A) causes the majority of food-borne outbreaks and has been observed to cause more severe symptoms with a higher mortality [4]. BoNTs are zinc proteases that cleave and inactivate cellular proteins essential for the release of the neurotransmitter acetylcholine. BoNT/A, -C, and -E cleave the peripheral plasma membrane protein Soluble N-ethylmaleimide-sensitive factor Attachment Protein of 25？kDA (SNAP-25); BoNT/B, -D, -F, and -G cleave synaptobrevin 2, also called as vesicle-associated membrane protein-2 (VAMP-2). In addition to cleaving SNAP-25, BoNT-C also cleaves the integral plasma membrane protein, syntaxin [1]. Cleavage of these substrates inhibits neuronal exocytosis [5]. The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) superfamily has become, since its discovery, the most intensively studied element of the protein machinery involved in intracellular trafficking. Currently, the only accepted assay with which to detect active Clostridium botulinum neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow
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