Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins

Abstract

Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

“…These assays expose a neuronal cell population to BoNT in defined media or buffer for a defined amount of time, followed by the analysis of an intracellular BoNT function such as SNARE protein cleavage or a block in neurotransmitter release or signal transduction. Comprehensive reviews on these methods are published elsewhere [89,[99][100][101]. Historically, these assays have used primary neuronal cultures derived from rodents or chickens, as well as continuous cell lines derived from cancer cells.…”

Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals

“…Muscarinic acetylcholine receptor: In two former projects, release of the reporter enzyme GLuc was stimulated by a high K + -depolarization buffer and entry of extracellular Ca 2+ into the reporter cell line [ 10 , 11 ]. One aim of this study was to analyze if GLuc can also be released by a Ca 2+ -increase from intracellular pools.…”

“…The last aim of the study was to analyze if the GLuc release from SIMA-hPOMC1-26-GLuc cells by high K + -depolarization can be inhibited by VGCC inhibitors rather than by BoNTs, which has previously been demonstrated [ 11 ]. Voltage dependent Ca 2+ -channels are a group of voltage-gated ion channels with a permeability for Ca 2+ -ions.…”

“…From these vesicles, GLuc was released upon depolarization of the cells into the cell culture supernatant together with neurotransmitters. In proof of principle, it was recently shown that the depolarization-dependent release was efficiently inhibited by BoNT/A and to a lesser extent by BoNT/B and tetanus toxin [ 10 , 11 ]. The goal of this study was to analyze whether this assay is also suitable to detect the activity of other neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release.…”

Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays

“…The effect of BoNTA that induces caspase-3- and caspase-7-dependent apoptotic processes, with inhibitory effects on the proliferation of the cell line T-47D, was used for evaluating the responses with 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) [18]. A new assay based on the inhibitory activity of BoNT on the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line, was suggested in order to measure the biological activity of the pharmaceutical preparations of BoNTA and bacterial neurotoxins [19]. Biological, immunological, and physicochemical methods used for Clostridium botulinum and its toxins were reviewed, mentioning their limitations and their usefulness for the detection and diagnostic [20].…”