Detection of black market follistatin 344

Abstract

Follistatin, a myostatin‐inhibiting protein, is prohibited according to chapter S4 of the “WADA 2019 List of Prohibited Substances and Methods”. While currently no approved pharmaceutical formulations of follistatin are available, follistatin can be bought on the black market. Most of the products are labeled “follistatin 344” (FS344), a few “follistatin 315”. A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only nine of the 17 tested products actually contained follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP‐2). Surprisingly, all nine products contained His‐tagged FS344 and a high degree of its oligomers. The detection method is based on immunomagnetic purification followed by SDS‐PAGE and Western blotting with a monoclonal anti‐His antibody. Alternatively, a monoclonal anti‐follistatin antibody can be used. For immunoprecipitation (IP), a polyclonal anti‐follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practically all currently available follistatin standards were investigated. The detection limit of the method for black market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 μL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His‐tags an unambiguous differentiation from endogenous follistatin is possible.

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…The class S4.4 of WADA's Prohibited List exhibits myostatin inhibitors including myostatin‐binding proteins such as, for example, follistatin, and while to date, no AAF with follistatin has been reported, and different follistatin preparations are advertised and sold by internet‐based providers. Hence, testing strategies are required, applicable to blood and/or urine as described by Reichel et al, 144 who studied the composition of available follistatin products and established analytical approaches employing immunopurification and electrophoretic analyses. Only nine out of 17 purchased follistatin products did indeed contain follistatin 315, and all of those contained a His‐tag supporting the differentiation of the natural peptide hormone from recombinantly produced and administered follistatin products.…”

“…The method was validated using a follistatin‐Fc fusion protein as model substance, and an LOD of 10 ng/ml was accomplished. While this approach was considered as ITP, confirmation analyses were conducted using the immunopurified serum (or plasma) extract for SDS‐PAGE and western blot detection similar to Reichel et al 144 …”

“…The method was validated using a follistatin-Fc fusion protein as model substance, and an LOD of 10 ng/ml was accomplished. While this approach was considered as ITP, confirmation analyses were conducted using the immunopurified serum (or plasma) extract for SDS-PAGE and western blot detection similar to Reichel et al 144 In addition to myostatin-binding proteins, also myostatin- Figure 2) and the consideration of complementary in-competition testing concerning stimulants using DBS 160 was suggested. , and yet the global regulatory status is complex, ranging from classifying CBD as a controlled substance to allowing for CBD products being sold as nutraceuticals.…”

Detection of black market follistatin 344

“…Several rows of product information in Tables 1 and 2 of the article by Reichel et al 1 were incorrectly captured during production of the article and information therefore appeared in the incorrect columns. The corrected Tables are provided here:…”

Myostatin inhibitory peptides in sports drug testing

“…Their misuse is prohibited both in-and out-of-competition, and they are listed in section S4 (Hormone and Metabolic Modulators) of the World Anti-Doping Agency's (WADA's) Prohibited List. 13 For several protein-based drug candidates such as the MSTN-neutralizing antibodies MYO-029 and Domagrozumab, 14,15 the anti-ActRII antibody Bimagrumab, 16 and inhibitors derived from FST, 17,18 doping control detection methods have been proactively developed. This is of utmost importance, as, even without clinical approval, reference material for such emerging drugs is often available for research purposes and/or distributed on the black market.…”