Updated protocols for the detection of Sotatercept and Luspatercept in human serum

• Published in: Drug testing and analysis 2018-11, Vol.10 (11-12), p.1708-1713
• Main Authors: Reichel, Christian, Gmeiner, Günter, Walpurgis, Katja, Thevis, Mario
• Format: Article
• Language: English
• Subjects: activin receptor; Activins - blood; Activins - isolation & purification; Antibodies, Immobilized - chemistry; Antigens; Biotinylation; Blotting, Western - methods; doping control; Drug testing; Electrophoresis, Polyacrylamide Gel - methods; Humans; Immunoglobulin Fc Fragments - blood; Immunoglobulin Fc Fragments - isolation & purification; Immunoprecipitation - methods; Luspatercept; Recombinant Fusion Proteins - blood; Recombinant Fusion Proteins - isolation & purification; SAR‐PAGE; Sotatercept
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.2500

We recently published two protocols for the detection of Sotatercept (ACE‐011, ACVR2A‐Fc) and Luspatercept (ACE‐536, ACVR2B‐Fc) in human serum. Both methods used covalently immobilized antibodies on agarose beads for immunoprecipitation and SAR‐PAGE/Western blotting for detection. Disadvantages were the relatively high amount of antibody required per sample (10 μg) and the need of a secondary antibody for the final detection. The updated protocols overcome these limitations by antigen–antibody complex formation in solution followed by capture of the complex with anti‐antibody‐coated magnetic beads. They also omit the secondary antibody incubation step by usage of biotinylated primary antibodies, which can be directly incubated with streptavidin‐HRP. Thus, the new protocols are faster, simpler, and cheaper and offer comparable sensitivities. We recently published SAR‐PAGE methods for the detection of Sotatercept (ACE‐011, ACVR2A‐Fc) and Luspatercept (ACE‐536, ACVR2B‐Fc) in human serum. The current article updates these protocols with respect to sample preparation and detection on Western blots. The new protocols are not only simpler and faster, but also cheaper. They can be easily adapted in case larger sample volumes than 50 μL need to be analyzed.