کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7233958 | 1470982 | 2013 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
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 55Â fM BoNT/A (1 LD50/ml) in 5Â min and 0.4Â fM (0.01 LD50/ml) in 5Â 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biosensors and Bioelectronics - Volume 49, 15 November 2013, Pages 276-281
Journal: Biosensors and Bioelectronics - Volume 49, 15 November 2013, Pages 276-281
نویسندگان
Christian Lévêque, Géraldine Ferracci, Yves Maulet, Chloé Grand-Masson, Marie-Pierre Blanchard, Michael Seagar, Oussama El Far,