Development of a Cell-Based Functional Assay for the Detection ofClostridium botulinumNeurotoxin Types A and E

Abstract

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.

Cellular bioelectric profiling: a novel method to differentially assess the in vitro toxicity of pesticides

“…For example, P. putida has been used for the detection of aromatic hydrocarbons and organophosphate nerve agents [24], while S. cerevisiae Y190 has been used for determining endocrine disruptor compounds [25]. In the same context, amperometric measurements have been used with E.coli, Salmonella typhimurium and Ralstonia eutropha in genotoxicity assays [26][27][28]. Clearly, using mammalian cells has considerable advantages over precaryotic ones, especially allowing for a more realistic reflection of the investigated toxic effects on biologic species of interest, including humans.…”

Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery

“…Secondly, they are easy to culture and generally not costly. Third, they can be easily manipulated to overexpress BoNT light chains and/or to generate reporter lines that can be used in FRET and/or FRAP based assays [34]. Based on these advantages, previously established cell-based BoNT assays utilized various continuous lines, including Neuro-2 a cells (mouse neuroblastoma) [35,36], P19 (mouse embryonic carcinoma) [37], SH-SY5Y (human neuroblastoma) [38], SK-N-SH (human neuroblastoma) [36], NT2 (human teratocarcinoma), BE(2)-M17 (human neuroblastoma) [39], and SiMa cells (human neuroblastoma) [40].…”

“…Based on these advantages, previously established cell-based BoNT assays utilized various continuous lines, including Neuro-2 a cells (mouse neuroblastoma) [35,36], P19 (mouse embryonic carcinoma) [37], SH-SY5Y (human neuroblastoma) [38], SK-N-SH (human neuroblastoma) [36], NT2 (human teratocarcinoma), BE(2)-M17 (human neuroblastoma) [39], and SiMa cells (human neuroblastoma) [40]. Similarly, PC12 cells (derived from rat pheochromocytoma) have been employed in FRET based approaches to measure the biological effects of BoNTs in cells [27,34]. Human carcinoma cells (N2) have also been utilized to measure vesicle exocytosis using fluorescent activity-dependent dyes [41].…”

“…Complex formation will not occur and no signal will be generated. The FRET principle has been utilized previously to create an in vitro assay for evaluating SNAP-25 proteolysis by BoNT/A via labeling both ends of a synthetic substrate [34]. However this type of FRET uses an artificial substrate analog that does not accurately reflect the physiological substrate within motor neurons.…”

Methods and difficulties in detection of Clostridium botulinum and its toxins

“…These assays provide a model for BoNT detection which requires all steps of the cellular intoxication including cell surface binding, endocytosis, translocation of the light chain (LC) of botulinum toxin into cellular cytosol, and enzymatic activity of the LC on SNARE substrates. Recently described NCB assays exceed the sensitivity of mouse bioassay (Basavanna et al 2013). Irrespective of the source of cells, all NCB assays require incubation of the cells with BoNTs for a defined time period, followed by the removal of the toxin and a quantitation of the endpoint for determining toxin activity.…”