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.
Type de document :
Article dans une revue
Biosensors and Bioelectronics, Elsevier, 2014, 57C, pp.207-212. 〈10.1016/j.bios.2014.02.015〉
Liste complète des métadonnées

http://www.hal.inserm.fr/inserm-00958576
Contributeur : Oussama El Far <>
Soumis le : mercredi 12 mars 2014 - 18:40:16
Dernière modification le : vendredi 8 juin 2018 - 13:40:02
Document(s) archivé(s) le : jeudi 12 juin 2014 - 12:11:11

Fichiers

Figure_3_Leveque_et_al.pdf
Fichiers produits par l'(les) auteur(s)

Identifiants

Collections

Citation

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〉

Partager

Métriques

Consultations de la notice

1045

Téléchargements de fichiers

1310