Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity

Abstract

Detection and quantification of low doses of botulinum toxin serotype A (BoNT/A) in medicinal preparations require precise and sensitive methods. With mounting pressure from governmental authorities to replace the mouse LD50 assay, interest in alternative methods such as the endopeptidase assay, quantifying the toxin active moiety, is growing. Using internal collision-induced fluorescence quenching, Pharmaleads produced peptides encompassing the SNAP-25 cleavage site: a 17-mer (PL63) and a 48-mer (PL50) reaching the previously identified α-exosite, with PL50 showing higher apparent affinity for BoNT/A. Peptide mapping experiments revealed that this increased affinity is mainly due to a connecting peptide sequence between the N-terminus of PL63 and the α-exosite, identifying a new cooperative exosite on BoNT/A. Other endopeptidase substrates available, including SNAPTide and BoTest A/E, are both based on fluorescent resonance energy transfer (FRET) technology. To compare these assays, their limits of detection and quantification were determined using light chain or 150-kDa BoNT/A. Detection limits of PL50 and BoTest were over 100 times better than those using SNAPtide in standard conditions. Although the BoTest possessed a detection limit around 0.2 pM for either BoNT/A form, its quantification limit (9.7 pM) using purified BoNT/A was 12 times inferior to PL50, estimated at 0.8 pM, suitable for medicinal preparation quantification.

Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate

“…Cognate substrate-based molecules are the main tools available to the researchers for the screening-based discovery of inhibitors. As has been shown by others 33 and us 13,23 , exosite-binding substrates can lead to a higher assay sensitivity for exosite inhibitors as compared to active-site substrates only. There is, however, a paucity of substrates that interact with exosites of ADAMs of interest.…”

Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology

“…While the Endopep-MS assay allows for the sensitive detection of all known serotypes with high confidence, the applicability of this approach is limited to expert laboratories due to the high level of expertise and expensive technical instrumentation needed. For a broader application, substrate peptides labelled with flanking fluorescence donor and quencher molecules have been designed to measure the catalytic activity of BoNT by Förster resonance energy transfer (FRET) 43–49 . Advantages of FRET-based approaches are their high sensitivity combined with a simple and fast assay protocol.…”

An Ultrasensitive Gold Nanoparticle-based Lateral Flow Test for the Detection of Active Botulinum Neurotoxin Type A

“…SNAPtide is a synthetic, commercially available, 13-amino acid peptide that contains the native SNAP-25 cleavage site for BoNT/A, the specifity of which has been confirmed extensively [16–18]. Hence, we only used BoNT/B-spiked and BoNT-negative sera to test the specifity of the LFTS in this study.…”