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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