A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A

authors

• Lévêque Christian
• Ferracci Géraldine
• Maulet Yves
• Grand-Masson Chloé
• Blanchard Marie-Pierre
• Seagar Michael
• El Far Oussama

keywords

• 4-2-hydroxyethyl-1-piperazine ethane sulfonic acid
• BSA
• BoNT
• DTT
• Dithiothreitol
• Endoprotease
• HBS
• HEPES-buffered saline
• Hepes
• LD50
• LOD
• LOQ
• Neo-epitope
• RU
• SNAP-25
• SPR
• Synaptosomal-associated protein 25
• Toxin sensor
• Botulinum neurotoxin
• Bovine serum albumin
• Limit of detection
• Limit of quantification
• Median lethal dose
• Resonance unit
• Surface plasmon resonance

abstract

Botulinum neurotoxin A (BoNT/A) induces muscle paralysis by enzymatically cleaving the presynaptic SNARE protein SNAP-25, which results in lasting inhibition of acetylcholine release at the neuromuscular junction. A rapid and sensitive in vitro assay for BoNT/A is required to replace the mouse lethality assay (LD50) in current use. We have developed a fully automated sensor to assay the endoprotease activity of BoNT/A. We produced monoclonal antibodies (mAbs) that recognize SNAP-25 neo-epitopes specifically generated by BoNT/A action. Recombinant SNAP-25 was coupled to the sensor surface of a surface plasmon resonance (SPR) system and samples containing BoNT/A were injected over the substrate sensor. Online substrate cleavage was monitored by measuring binding of mAb10F12 to a SNAP-25 neo-epitope. The SNAP-25-chip assay was toxin serotype-specific and detected 55 fM BoNT/A (1 LD50/ml) in 5 min and 0.4 fM (0.01 LD50/ml) in 5h. Time-course and dose-response curves were linear, yielding a limit of quantification of 0.03 LD50/ml. This label-free method is 100 times more sensitive than the mouse assay, potentially providing rapid read-out of small amounts of toxin for environmental surveillance and the quality control of pharmaceutical preparations.

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