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. 2024 Jul 12;6:1407409. doi: 10.3389/fmedt.2024.1407409

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© 2024 Kasim, Chillon, Eleftheriadou, Rijntjes, Minich, Zechmann and Schomburg.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Establishment and verification of the new method and characterization of stability of ghrelin-aAb in serum. (A) A detection assay was designed, consisting of a secreted alkaline phosphatase (SEAP) in frame to human ghrelin as one fusion protein, to be used as bait for the autoantibodies (Y-symbol, ghrelin-specific immunoglobulin highlighted in red) that become enriched by protein A-mediated immunoprecipitation. Detection is achieved by incubation with a chemiluminescent substrate (CSPD), and relative light units (RLU) are recorded by a luminometer. After the establishment of stable clones expressing SEAP-ghrelin fusion protein, the signal detection method and certain matrix characteristics were characterized. (B) Increasing signal strength (RLU) was observed with increasing amount of commercial anti-ghrelin antibody in the new assay, supporting its general suitability to detect and quantify ghrelin-aAb. (B) Signal stability of positive samples was tested by incubation for 5 days and 10 days at room temperature or 4°C. The comparison to the initial signal strength indicates high stability of ghrelin-aAb in serum under these conditions.