title: 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 5 fM (0.1LD50/ml serum) was obtained. However, this configuration required prior immunoprecipitation of BoNT/A. A sensitivity of 0.5 fM 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 50-100 µl 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.
authors.affiliation: INSERM, UMR_S 1072, 13015 Marseille, France; Aix-Marseille Université, 13015 Marseille, France. Electronic address: christian.leveque@univ-amu.fr.
authors.forename: Christian
authors.initials: C
authors.lastname: Lévêque
authors.affiliation: CNRS, UMR 7286, Plate-Forme de Recherche en Neurosciences PFRN, 13015 Marseille, France; Aix-Marseille Université, 13015 Marseille, France.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Ferracci
authors.affiliation: INSERM, UMR_S 1072, 13015 Marseille, France; Aix-Marseille Université, 13015 Marseille, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Maulet
authors.affiliation: CNR Anaérobies et botulisme, Unité Bactéries anaérobies et toxines. Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Mazuet
authors.affiliation: CNR Anaérobies et botulisme, Unité Bactéries anaérobies et toxines. Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Popoff
authors.affiliation: INSERM, UMR_S 1072, 13015 Marseille, France; Aix-Marseille Université, 13015 Marseille, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Seagar
authors.affiliation: INSERM, UMR_S 1072, 13015 Marseille, France; Aix-Marseille Université, 13015 Marseille, France.
authors.forename: Oussama
authors.initials: O
authors.lastname: El Far
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D019274:Botulinum Toxins, Type A / Q000097:blood* / Q000378:metabolism; D001906:Botulism / Q000097:blood* / Q000175:diagnosis / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D051379:Mice; D010447:Peptide Hydrolases / Q000097:blood / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D010447:Peptide Hydrolases; D019274:Botulinum Toxins, Type A
keywords: Botulinum neurotoxin; Serum detection; Surface plasmon resonance; Toxin sensor; Type A botulism