Detection of Sotatercept (ACE-011, ActRIIA-IgGI) in human urine and blood – Protocols for initial and confirmatory doping testing
Aside from human endogenous erythropoietin (EPO), its recombinant analogues (e.g. Epoetins alfa/beta/delta/omega, Darbepoetin alfa) and EPO-mimetics (e.g. Peginesatide), which stimulate erythropoieses through the EPO-receptor pathway, formation of erythrocytes can also be stimulated via the so called €activin-receptor type IIA signal transduction pathwayÅ: as soon as ligands, which interact with activin-receptor type IIA (€ActRIIA ligandsÅ) are removed by so-called ActRIIA ligand traps in a targeted way, erythropoiesis is also stimulated. ActRIIA ligands belong in particular to the transforming growth factor-beta (TGF-‚) superfamily (e.g. activin).
Sotatercept (ACE-011, ActRIIA-IgGI), a fusion protein consisting of the extracellular domain of ActRIIA receptor and the Fc-part of human immunoglobulin G1 (IgG1), is capable of acting as ActRIIA ligand trap, and was primarily developed as pharmaceutical for enhancing bone mineralisation in order to revert osteoporosis. However, aside from increasing bone mineral density, it was discovered that Sotatercept also stimulates erythropoiesis in a dose dependant manner leading to an increase in red blood cell counts. Sotatercept is currently intensely investigated in several phase II clinical trials.
Two methods for the detection of Sotatercept in serum and urine samples were successfully developed. One method uses covalent immobilization of the capture antibody on agarose beads. It requires 10 µg of a polyclonal goat anti-ACVR2A antibody per sample (AF340 from R&D Systems) but needs double-blotting in case multiplexed detection of Sotatercept together with epoetins is necessary. The other method applies commercial magnetic beads coated with an antibody (sheep anti-rabbit) directed against the capture antibody. After incubation of the serum samples, the complex consisting of Soatercept and the capture antibody (a polyclonal rabbit antibody, 10257-T52 from Sino Biological) is bound by the anti-rabbit antibodies on the beads. After Western blotting, Soatercept is detected with the biotinylated version of the capture antibody of the other method (BAF340 from R&D Systems) and streptavidin-HRP. It omits incubation with a secondary antibody and thus is simpler, faster, and cheaper (only ca. 1.5 µg of capture antibody are required) The second protocol is compatible with single-blotting if the biotinylated clone AE7A5 antibody is used for the simultaneous detection of epoetins.