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

作者：Leveque Christian^*; Ferracci Geraldine; Maulet Yves; Grand Masson Chloe; Blanchard Marie Pierre; Seagar Michael; El Far Oussama

来源：Biosensors and Bioelectronics, 2013, 49: 276-281.

DOI：10.1016/j.bios.2013.05.032

• Botulinum neurotoxin
• SNAP-25
• Surface plasmon resonance
• Toxin sensor
• Endoprotease
• Neo-epitope

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 neoepitope. 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 5 h. 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.

• 出版日期2013-11-15