Abstract
Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A’s location, distribution, and movement within a cell. Due to BoNT/A’s high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25197) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25197, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25197 rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25197 in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25197-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.
Highlights
Botulinum neurotoxin type A (BoNT/A) causes transient muscle paralysis through presynaptic blockade of acetylcholine release at the neuromuscular junction (NMJ)
We introduce several recombinant monoclonal antibodies (rMAb) that were developed in-house against SNAP25197 and compared their immuno-reactive signal against that of commercial antibodies using a variety of different methods (Table 2)
Within the field of botulinum neurotoxins, cleavage site-specific antibodies can help detect the activity of minute quantities of BoNT light-chain that may otherwise be very difficult to perceive
Summary
Botulinum neurotoxin type A (BoNT/A) causes transient muscle paralysis through presynaptic blockade of acetylcholine release at the neuromuscular junction (NMJ). While the general mechanism of action (MoA) for BoNT/A at the presynaptic nerve terminal has been well established [10,11], there are still many unanswered questions regarding the intracellular trafficking patterns and general “life-cycle” of the toxin. Resolving these questions partly depends on the ability to precisely detect the toxin’s location, distribution, and movement within a cell.
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