Simultaneous detection method of muscle developing monoclonal antibodies with using LC-MS/MS from Dried Blood Spots

Abstract

Muscle wasting is linked to diseases like muscular dystrophy, type 2 diabetes, and cardiovascular issues. Current treatments mainly focus on slowing muscle degeneration through nutrition. An emerging strategy targets the TGF-β superfamily's receptor signaling, including myostatin, activin A, and GDF-11, which inhibit muscle growth. Blocking the myostatin receptor pathway can increase muscle mass. However, using inhibitors for athletic performance enhancement is prohibited by World Anti-Doping Agency(WADA). A multiplex detection method was created to identify prohibited monoclonal antibodies in doping, focusing on those targeting the TGF-β superfamily: Trevogrumab, Landogrozumab, Domagrozumab, and Bimagrumab. Dried blood spot cards were used, and antibodies were extracted from DBS and purified using protein G magnetic beads. Dulaglutide served as an internal standard. The method involved simultaneous detection of antibodies and Dulaglutide, followed by LC-MSMS analysis. Validation included specificity, selectivity, linearity, carryover, recovery, and matrix effect. All target antibody concentrations were below the limit of detection (LOD), which is less than 1？g/ml. Despite lacking clinical approval, detecting anti-TGF-β superfamily antibodies is crucial for doping control, applicable to other protein therapeutics. Furthermore, the method using dried blood spot cards offers a fast, simple, safe, and minimally invasive process with stability for analysts and athletes.