Abstract
The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins.
Highlights
The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance
Mice were immunized using an eight residue peptide corresponding to the new C-terminal domain of Synaptosomal-associated protein of 25 kDa (SNAP-25) produced by the proteolytic activity of Botulinum neurotoxins (BoNTs)/E (Fig. 1a)
One clone was selected and characterized as follows: upon injection in the mobile phase, strong binding to immobilized HisSNAP25 previously cleaved by BoNT/E was measured, whereas no binding was detected either to an irrelevant control protein (GST) or to uncleaved His-SNAP25 (Fig. 1b)
Summary
The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. BoNT’s found in human sera are in the low fmolar concentration range, specific receptors localized in the peripheral nervous system concentrate the toxin, mediate its neuronal internalization and the consequent disabling of synaptic transmission[1] These potent neurotoxins are considered as one of the most toxic bioweapons constituting a threat for the public[2]. The decision to use serotherapy is often taken on the basis of clinical symptoms and the most direct way to subsequently confirm diagnosis is to identify the BoNT type in the patient’s serum and/or stool using the standard mouse median lethal dose LD50 method[4] The specificity of this in vivo assay lies in the absence of symptoms when specific anti-BoNT antibodies are co-injected with the experimental sample
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