title: A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A.
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.
authors.affiliation: INSERM, UMR_S 1072, 13015 Marseille, France; Aix-Marseille Université, 13015 Marseille, France. christian.leveque@univ-amu.fr
authors.forename: Christian
authors.initials: C
authors.lastname: Lévêque
authors.forename: Géraldine
authors.initials: G
authors.lastname: Ferracci
authors.forename: Yves
authors.initials: Y
authors.lastname: Maulet
authors.forename: Chloé
authors.initials: C
authors.lastname: Grand-Masson
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Blanchard
authors.forename: Michael
authors.initials: M
authors.lastname: Seagar
authors.forename: Oussama
authors.initials: O
authors.lastname: El Far
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000032:analysis* / Q000378:metabolism*; D001906:Botulism / Q000382:microbiology; D003014:Clostridium botulinum / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D057230:Limit of Detection; D051379:Mice; D009498:Neurotoxins / Q000032:analysis* / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D055767:Immobilized Proteins; D009498:Neurotoxins; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D010447:Peptide Hydrolases; D019274:Botulinum Toxins, Type A
keywords: 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid; BSA; BoNT; Botulinum neurotoxin; DTT; Dithiothreitol; Endoprotease; HBS; HEPES-buffered saline; Hepes; LD(50); LOD; LOQ; Neo-epitope; RU; SNAP-25; SPR; Surface plasmon resonance; 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