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Proteomics Clin Appl. 2016 Feb;10(2):195-205. doi: 10.1002/prca.201500043. Epub 2015 Oct 23.

Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

Author information

1
Center for Preventive Doping Research/Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.
2
European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany.

Abstract

PURPOSE:

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.

EXPERIMENTAL DESIGN:

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 AND CLINICAL RELEVANCE:

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.

KEYWORDS:

Affinity purification; Doping; Myostatin; Therapeutic antibodies; Western blotting

PMID:
26223570
DOI:
10.1002/prca.201500043
[Indexed for MEDLINE]

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