title: Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.
abstract: OBJECTIVE
  Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches.
  
  
  METHODS
  To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery.
  
  
  RESULTS
  The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%.
  
  
  CONCLUSIONS
  Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples.
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Walpurgis
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
authors.affiliation: Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.|European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004300:Doping in Sports; D006801:Humans; D055435:Myostatin / Q000037:antagonists & inhibitors* / Q000276:immunology*; D058573:Performance-Enhancing Substances / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D057134:Antibodies, Neutralizing; C525911:MSTN protein, human; D055435:Myostatin; D058573:Performance-Enhancing Substances
keywords: Affinity purification; Doping; Myostatin; Therapeutic antibodies; Western blotting