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




A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A - Download this document for free, or read online. Document in PDF available to download.

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

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.

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





Author: Christian Lévêque - Géraldine Ferracci - Yves Maulet - Chloé Grand-Masson - Marie-Pierre Blanchard - Michael Seagar - Oussama

Source: https://hal.archives-ouvertes.fr/



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