Development of Cell-Based Assays to Measure Botulinum Neurotoxin Serotype A Activity Using Cleavage-Sensitive Antibodies

Abstract

Botulinum neurotoxins (BoNTs) are zinc-metalloproteases that cleave components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complex, inhibiting acetylcholine release into neuromuscular junctions, resulting in flaccid paralysis and eventual death. The potential for the malicious misuse of these toxins as bioweapons has created an urgent need to develop effective therapeutic countermeasures. Robust cell-based assays will be essential for lead identification and the optimization of therapeutic candidates. In this study, the authors developed novel BoNT serotype A (BoNT/A) cleavage-sensitive (BACS) antibodies that only interact with full-length SNAP-25 (synaptosomal-associated protein of 25 kDa), the molecular target of the BoNT/A serotype. These antibodies exhibit high specificity for full-length SNAP-25, allowing the BoNT/A-mediated proteolysis of this protein to be measured in diverse assay formats, including several variations of enzyme-linked immunosorbent assay and multiple immunofluorescence methods. Assays built around the BACS antibodies displayed excellent sensitivity, had excellent reproducibility, and were amenable to multiwell formats. Importantly, these assays provided novel methods for evaluating BoNT/A activity in cellular models of intoxication and allowed for the high-throughput evaluation of experimental compounds. Botulinum neurotoxins (BoNTs) are zinc-metalloproteases that cleave components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complex, inhibiting acetylcholine release into neuromuscular junctions, resulting in flaccid paralysis and eventual death. The potential for the malicious misuse of these toxins as bioweapons has created an urgent need to develop effective therapeutic countermeasures. Robust cell-based assays will be essential for lead identification and the optimization of therapeutic candidates. In this study, the authors developed novel BoNT serotype A (BoNT/A) cleavage-sensitive (BACS) antibodies that only interact with full-length SNAP-25 (synaptosomal-associated protein of 25 kDa), the molecular target of the BoNT/A serotype. These antibodies exhibit high specificity for full-length SNAP-25, allowing the BoNT/A-mediated proteolysis of this protein to be measured in diverse assay formats, including several variations of enzyme-linked immunosorbent assay and multiple immunofluorescence methods. Assays built around the BACS antibodies displayed excellent sensitivity, had excellent reproducibility, and were amenable to multiwell formats. Importantly, these assays provided novel methods for evaluating BoNT/A activity in cellular models of intoxication and allowed for the high-throughput evaluation of experimental compounds.