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Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism.

Abstract : : Botulinum neurotoxin A (BoNT/A) has intrinsic endoprotease activity specific for SNAP-25, a key protein for presynaptic neurotransmitter release. The inactivation of SNAP-25 by BoNT/A underlies botulism, a rare but potentially fatal disease. There is a crucial need for a rapid and sensitive in vitro serological test for BoNT/A to replace the current in vivo mouse bioassay. Cleavage of SNAP-25 by BoNT/A generates neo-epitopes which can be detected by binding of a monoclonal antibody (mAb10F12) and thus measured by surface plasmon resonance (SPR). We have explored two SPR assay formats, with either mAb10F12 or His6-SNAP-25 coupled to the biosensor chip. When BoNT/A was incubated with SNAP-25 in solution and the reaction products were captured on a mAb-coated chip, a sensitivity of 5fM (0.1LD50/ml serum) was obtained. However, this configuration required prior immunoprecipitation of BoNT/A. A sensitivity of 0.5fM in 10% serum (0.1 LD50/ml serum) was attained when SNAP-25 was coupled directly to the chip, followed by sequential injection of BoNT/A samples and mAb10F12 into the flow system to achieve on-chip cleavage and detection, respectively. This latter format detected BoNT/A endoprotease activity in 50-100µl serum samples from all patients (11/11) with type A botulism within 5h. No false positives occurred in sera from healthy subjects or patients with other neurological diseases. The automated chip-based procedure has excellent specificity and sensitivity, with significant advantages over the mouse bioassay in terms of rapidity, required sample volume and animal ethics.
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https://www.hal.inserm.fr/inserm-00958576
Contributor : Oussama El Far Connect in order to contact the contributor
Submitted on : Wednesday, March 12, 2014 - 6:40:16 PM
Last modification on : Tuesday, October 19, 2021 - 10:59:32 PM
Long-term archiving on: : Thursday, June 12, 2014 - 12:11:11 PM

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Christian Lévêque, Géraldine Ferracci, Yves Maulet, Christelle Mazuet, Michel Popoff, et al.. Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism.. Biosensors and Bioelectronics, Elsevier, 2014, 57C, pp.207-212. ⟨10.1016/j.bios.2014.02.015⟩. ⟨inserm-00958576⟩

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