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

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 neoepitopes which can be detected by binding of a monoclonal antibody (mAbl0F12) and thus measured by surface plasmon resonance (SPR). We have explored two SPR assay formats, with either mAbl0F12 or His5-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 (0.1LID(50)/m1 serum) was obtained. However, this configuration required prior immunoprecipitation of BoNT/A. A sensitivity of 0.5 fM in 10% serum (0.1 LD50/m1 serum) was attained when SNAP-25 was coupled directly to the chip, followed by sequential injection of BoNT/A samples and mAbl0F12 into the flow system to achieve on-chip cleavage and detection, respectively. This latter format detected BoNT/A endoprotease activity in 50-100 mu 1 serum samples from all patients (11/11) with type A botulism within 5 h. No false positives occurred in sera from healthy subjects or patients with other neurological diseases. The automated chipbased procedure has excellent specificity and sensitivity, with significant advantages over the mouse bioassay in terms of rapidity, required sample volume and animal ethics. (C) 2014 Elsevier B.V. All rights reserved.