Detection of activin receptor type IIA and IIB‐Fc fusion proteins by automated capillary immunoassay

Abstract

Activin receptor type IIA and type IIB fusion protein have been designed to sequester circulating molecules of the transforming growth factor‐β (TGF‐β) superfamily and inactivate their actions. Members of this superfamily have been reported as essential regulators of erythropoiesis by triggering the formation of activated ternary complexes containing different combinations of type I and type II receptors, which can limit RBC production by accelerating erythroid differentiation and inhibiting erythroid progenitor expansion. The recent approval of Luspatercept for the treatment of anemia associated to transfusion‐dependent MDS and Beta‐thalassemia in afflicted patients means that it can now pose a real threat of being abused in sport for its ability to stimulate erythropoiesis. Several methods for the detection of these molecules in blood have been proposed for the purpose of sport antidoping control. Here we propose the detection of the ActRIIA‐Fc and ActRIIB‐Fc fusion proteins by automated capillary Western immunoassay (Simple Western). The use of these immunoassays for the detection of protein targets has become widespread in the recent years. The work presented here demonstrates that this methodology enables a versatile, rapid, and sensitive detection of activin ligand traps in blood samples: plasma, serum, or dried blood spots (DBS). Preliminary results indicate that detection in urine samples is also possible. The option to use different antibodies allows the possibility to use this method as an initial testing procedure as well as a confirmation procedure. Finally, results coming from an administration study confirm that the method is suitable for routine analysis.

The 40th Manfred Donike workshop on doping analysis

“…These methods facilitated serum steroid profiling 10 and proved applicable also to DBS, 11 where considerable advantages especially regarding steroid ester stabilities were documented. 12 Detecting the manipulation of erythropoiesis with newly approved drugs such as luspatercept has been the subject of in-depth investigations, and two analytical approaches utilizing either an automated capillary immunoassay 13 or gel-based electrophoretic methods 14 were shown to be fit-for-purpose and compatible with serum, DBS, and urine analyses. In a proof-of-concept study, a therapeutic dosing (two injections within 3 weeks) was shown to be traceable for at least 7 weeks by direct test methods, while hematological markers were affected only marginally.…”