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

Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism. - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

* Corresponding author 1 UNIS - Unité de Neurobiologie des canaux Ioniques et de la Synapse 2 CRN2M - Centre de recherche en neurobiologie - neurophysiologie de Marseille 3 Bactéries anaérobies et Toxines

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.

Keywords : botulinum neurotoxin serum detection surface plasmon resonance toxin sensor type A botulism

Autor: Christian Lévêque - Géraldine Ferracci - Yves Maulet - Christelle Mazuet - Michel Popoff - Michael Seagar - Oussama El Far -



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