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

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• 作者：Christian L¨¦v¨ºque ; G¨¦raldine Ferracci ; Yves Maulet ; Chlo¨¦ Gr ; -Masson ; Marie-Pierre Blanchard ; Michael Seagar ; Oussama El Far
• 关键词：BoNT ; botulinum neurotoxin ; SPR ; surface plasmon resonance ; RU ; resonance unit ; BSA ; bovine serum albumin ; LD50 ; median lethal dose ; Hepes ; 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid ; SNAP-25 ; Synaptosomal-associated protein 25 ; DTT ; Dithiothrei
• 刊名：Biosensors and Bioelectronics
• 出版年：2013
• 出版时间：15 November, 2013
• 年：2013
• 卷：49
• 期：Complete
• 页码：276-281
• 全文大小：1187 K

文摘

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 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.