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. 2021 Aug 4;11:15791. doi: 10.1038/s41598-021-95305-y

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Schematic representation of the AMA Assay. Step 1—amidation: calibrators, controls and samples are dispensed in double determination as indicated (yellow) into a non-binding 96-well plate. The reaction is started by addition of reaction buffer to calibrators, controls and samples (green) and a t = 0 min time-point is generated immediately by transferring equal volumes of each double determination into EDTA prefilled wells (red). The generated t = 0 min time-points are single determinations. After incubation at 37 °C for 40 min an inactivation of remaining samples with EDTA is performed. Step 2—product quantification: following 37 °C incubation and inactivation, all samples are transferred into the sphingotest bio-ADM Assay 96-well immunoluminometric assay (ILMA) plate. The bio-ADM assay is processed according to manufacturer’s specifications. Both, ADM-Gly and bio-ADM are bound by the solid-phase antibody (a). The MACN labelled tracer-antibody (b) specifically recognizes the c-terminally amidated bio-ADM and does not react with ADM-Gly. Following incubation, unbound tracer-antibody is washed away and bio-ADM specific luminescence signals are detected using a standard plate luminometer. The figure was created with Microsoft PowerPoint 2016, version 2105.