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. 2019 Apr 2;9:5531. doi: 10.1038/s41598-019-41722-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Schematic illustration of the Luminex duplex-assay working principle. (a) Paramagnetic beads coupled to monoclonal anti-BoNT antibodies are added to a toxin containing sample for toxin enrichment. For the three duplex-assays paramagnetic beads containing either anti-A and anti-B, or anti-C and anti-D, or anti-E and anti-F are mixed to enable simultaneous enrichment of two different serotypes in one sample. (b) SNAP-25 and VAMP-2 are coupled to two different regions of Luminex microspheres. Luminex microspheres are paramagnetic beads carrying carboxyl groups to enable coupling of molecules via primary amine groups. Each Luminex bead region carries a distinct ratio of two different fluorescent dyes. Hereby up to 100 colour-coded bead regions can be discerned via their fluorescence emission pattern after excitation. Different BoNT serotypes cleave SNAP-25 or VAMP-2 at distinct, individual sites. The resulting serotype-specific neoepitopes can be detected by Neo-mAbs (Neo-A to Neo-F). The differently coded microspheres for SNAP-25 and VAMP-2 allow for the simultaneous detection of one SNAP- and one VAMP-specific BoNT. (c) Workflow of the duplex-assay exemplarily shown for the BoNT/A + BoNT/B duplex assay with BoNT/A contained in the sample: After enrichment captured toxin is added to VAMP-2 and SNAP-25 coupled Luminex beads where in this case only SNAP-25 is cleaved at the BoNT/A-specific cleavage site. After adding a mixture of Neo-mAbs specific for BoNT/A and BoNT/B, only BoNT/A-specific Neo-mAbs bind to cleaved SNAP-25 immobilised to the colour-coded Luminex-beads enabling specific discrimination between one SNAP-25 and one VAMP-2 cleaving serotype in one assay. The fluorescence-intensity correlates with the amount of cleaved substrate and therefore indicates the concentration of BoNT in the sample.