Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology

Abstract

Botulinum neurotoxins (BoNTs) are the most potent toxins known and cause the life threatening disease botulism. Sensitive and broad detection is extremely challenging due to the toxins’ high potency and molecular heterogeneity with several serotypes and more than 40 subtypes. The toxicity of BoNT is mediated by enzymatic cleavage of different synaptic proteins involved in neurotransmitter release at serotype-specific cleavage sites. Hence, active BoNTs can be monitored and distinguished in vitro by detecting their substrate cleavage products. In this work, we developed a comprehensive panel of monoclonal neoepitope antibodies (Neo-mAbs) highly specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. The Neo-mAbs were implemented in a set of three enzymatic assays for the simultaneous detection of two BoNT serotypes each by monitoring substrate cleavage on colour-coded magnetic Luminex-beads. For the first time, all relevant serotypes could be detected in parallel by a routine in vitro activity assay in spiked serum and food samples yielding excellent detection limits in the range of the mouse bioassay or better (0.3–80 pg/mL). Therefore, this work represents a major step towards the replacement of the mouse bioassay for botulism diagnostics.

Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays

“…Botulinum neurotoxins (BoNTs) are a group of bacterial exotoxins, which are the most potent toxins known to occur naturally. There are seven well-studied serotypes in addition to mosaic forms and numerous subtypes, which are produced by bacteria of the genus Clostridium [1]. All serotypes are produced from a single polypeptide precursor cleaved into a small and a large subunit.…”

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A–F Employing Taguchi Design-of-Experiments

“…Hereby, our optimized cleavage buffers are instrumental to the improvement of cleavage based assays for BoNT detection. In our recent work, the consensus buffer was successfully implemented in a neoepitope-based suspension array for the functional detection of botulinum neurotoxin serotypes A–F [52]. Finally, we demonstrated that the Taguchi DoE method is a straightforward tool that is transferable to optimize other multi-parameter enzymatic reactions.…”

“…Förster resonance energy transfer (FRET)-based endopeptidase-assays measure the cleavage of serotype-specific peptide substrates labelled with fluorescent reporter molecules by a change in fluorescent intensity [33,34,35,36,37,38,39,40,41]. Another approach relies on the immunodetection of cleavage products by neo-epitope-specific antibodies that recognize only the cleaved and not the uncleaved substrate [42,43,44,45,46,47,48,49,50,51,52]. Furthermore, cleavage products can be detected by mass spectrometry, where the cleavage of serotype-specific peptide substrates is analyzed by monitoring unique peptide products [53,54].…”

Multiplex Immunoassay Techniques for On-Site Detection of Security Sensitive Toxins

“…Therefore, the authors developed a panel of monoclonal neoepitope antibodies specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. These antibodies were applied in three duplex assays to discriminate BoNT/A to /F achieving LODs in the range of 0.3–80 pg/mL with a total assay time of approx. 21 h [ 123 ]. Particularly, the catalytic cleavage reaction required an incubation time of 18 h to achieve LODs in the pg/mL range.…”