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

Christian Lévêque (1) , Géraldine Ferracci (2) , Yves Maulet (1) , Chloé Grand-Masson (2) , Marie-Pierre Blanchard (2) , Michael Seagar (1) , Oussama El Far (1)

1 UNIS - Inserm U1072 - Unité de Neurobiologie des canaux Ioniques et de la Synapse
2 CRN2M - Centre de recherche en neurobiologie - neurophysiologie de Marseille

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.

Mots clés: 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid; BSA; BoNT; DTT; Dithiothreitol; Endoprotease; HBS; HEPES-buffered saline; Hepes; LD(50); 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

Domaines: Biochimie [q-bio.BM]; Biotechnologies; Biotechnologie

Christian Lévêque, Géraldine Ferracci, Yves Maulet, Chloé Grand-Masson, Marie-Pierre Blanchard, et al.. A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A. Biosensors and Bioelectronics, 2013, 49, pp.276-281. ⟨10.1016/j.bios.2013.05.032⟩. ⟨hal-00958814⟩