Antibody‐based strategies for the detection of Luspatercept (ACE‐536) in human serum

Luspatercept (ACE‐536, ACVR2B‐Fc), a fusion protein consisting of the extracellular domain of ActRIIB receptor and the Fc‐part of human immunoglobulin G1 (IgG1), is currently under clinical development (Phase III). It stimulates the formation of red blood cells and hence may be misused by athletes for doping purposes in the future. Several antibody‐based strategies for the detection of Luspatercept and other ACVR2B‐Fc fusion proteins in human serum were evaluated (ELISA; IEF‐, SDS‐, and SAR‐PAGE followed by Western blotting; immunoprecipitation). Two methods led to useful results: a commercial “soluble” ACTR‐IIB ELISA, which also detected Luspatercept and other ACVR2B‐Fc's, but showed no cross‐reactivity with Sotatercept/ACVR2A‐Fc's. The ELISA might be applied as fast screening tool (100 μL serum; limit of detection (LOD) ca 15.6 ng/mL). The second method uses a polyclonal ACVR2B‐antibody for immunoprecipitation followed by SAR‐PAGE and Western blotting with a monoclonal detection antibody (50 μL serum; LOD ca 1.0 ng/mL). It can be used for initial as well as for confirmatory testing. Due to the high doses (mg/kg) and long serum half‐life of Luspatercept, both strategies may be useful in anti‐doping control in the future. Copyright © 2017 John Wiley & Sons, Ltd. Several immunological strategies were tested in order to develop a detection method for Luspatercept and other ACVR2B‐Fc fusion proteins in serum samples (ELISA, IEF‐PAGE, SDS/SAR‐PAGE plus Western blotting). While Luspatercept can be determined in human serum with a commercial ACVR2B‐ELISA (LOD ca 15.6 ng/mL; 100 μL serum), the developed SAR‐PAGE method is more sensitive and allows the detection in only 50 μL serum down to ca 1.0 ng/mL. Considering the long serum half‐life (15–16 days) and high doses used for stimulating erythropoiesis, both the ELISA‐ and SAR‐PAGE methods will be able to detect Luspatercept misuse over a period of several weeks.