Sensitivity and specificity of BoNT detection by three Luminex duplex-assays. As indicated in Fig. 3, the different serotypes BoNT/A to F were diluted in BSA/PBS and enriched with monoclonal anti-BoNT antibodies coupled to paramagnetic beads (duplex enrichment: A + B, C + D, E + F). Captured toxin was mixed with VAMP-2 and SNAP-25 coupled to Luminex microspheres for substrate cleavage and cleavage products were subsequently detected by a mixture of Neo-mAbs targeting the cleavage sites of BoNT/A and B (a), BoNT/C and D (b), or BoNT/E and F (c). Results from two independent experiments with each repeat performed in technical duplicates are shown. (n = 4; mean ± SD; MFI = Median fluorescent intensity). Inserts show cut-off values (horizontal dashed lines) used to calculate the limits of detection (vertical dashed lines) zoomed in on the relevant areas of the graphs.