Detection of Sotatercept (ACE-011) in human serum by SAR-PAGE and western single blotting

Christian Reichel; Letizia Farmer; Günter Gmeiner; Katja Walpurgis; Mario Thevis

doi:10.1002/dta.2346

Detection of Sotatercept (ACE-011) in human serum by SAR-PAGE and western single blotting

Christian Reichel^*, Letizia Farmer, Günter Gmeiner, Katja Walpurgis, Mario Thevis

^*Corresponding author for this work

Publication: Contribution to journal › Journal articles › Research › peer-review

Abstract

A method for the detection of Sotatercept (ACE-011, ACVR2A-Fc) in human serum is presented. The method is a modification of a recently published protocol for Luspatercept (ACE-536, ACVR2B-Fc), another erythropoiesis stimulating fusion protein. Out of 27 tested antibodies against either the extracellular domain of ACVR2A or the full-length protein, only 4 antibodies bound strongly enough to Sotatercept for usage with immunoprecipitation followed by SAR-PAGE and Western single blotting. The adapted protocol allows the detection of 0.1 ng/mL Sotatercept in just 50 μL human serum. None of the 3 commercial ACVR2-ELISAs was able to detect Sotatercept and the 2 tested surrogate proteins, even in the μg/mL range. As for Luspatercept, only IPG-IEF/2D-PAGE generated discrete isoforms. Due to the long serum half-life, the SAR-PAGE method will be able to detect Sotatercept for several weeks and will be very useful in doping testing.

Original language	English
Journal	Drug testing and analysis
Volume	10
Issue number	6
Pages (from-to)	927-937
Number of pages	11
ISSN	1942-7603
DOIs	https://doi.org/10.1002/dta.2346
Publication status	Published - 2018

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title = "Detection of Sotatercept (ACE-011) in human serum by SAR-PAGE and western single blotting",

abstract = "A method for the detection of Sotatercept (ACE-011, ACVR2A-Fc) in human serum is presented. The method is a modification of a recently published protocol for Luspatercept (ACE-536, ACVR2B-Fc), another erythropoiesis stimulating fusion protein. Out of 27 tested antibodies against either the extracellular domain of ACVR2A or the full-length protein, only 4 antibodies bound strongly enough to Sotatercept for usage with immunoprecipitation followed by SAR-PAGE and Western single blotting. The adapted protocol allows the detection of 0.1 ng/mL Sotatercept in just 50 μL human serum. None of the 3 commercial ACVR2-ELISAs was able to detect Sotatercept and the 2 tested surrogate proteins, even in the μg/mL range. As for Luspatercept, only IPG-IEF/2D-PAGE generated discrete isoforms. Due to the long serum half-life, the SAR-PAGE method will be able to detect Sotatercept for several weeks and will be very useful in doping testing.",

keywords = "Journal Article",

author = "Christian Reichel and Letizia Farmer and G{\"u}nter Gmeiner and Katja Walpurgis and Mario Thevis",

note = "Copyright {\textcopyright} 2017 John Wiley & Sons, Ltd.",

year = "2018",

doi = "10.1002/dta.2346",

language = "English",

volume = "10",

pages = "927--937",

journal = "Drug testing and analysis",

issn = "1942-7603",

publisher = "John Wiley & Sons, Ltd",

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TY - JOUR

T1 - Detection of Sotatercept (ACE-011) in human serum by SAR-PAGE and western single blotting

AU - Reichel, Christian

AU - Farmer, Letizia

AU - Gmeiner, Günter

AU - Walpurgis, Katja

AU - Thevis, Mario

PY - 2018

Y1 - 2018

N2 - A method for the detection of Sotatercept (ACE-011, ACVR2A-Fc) in human serum is presented. The method is a modification of a recently published protocol for Luspatercept (ACE-536, ACVR2B-Fc), another erythropoiesis stimulating fusion protein. Out of 27 tested antibodies against either the extracellular domain of ACVR2A or the full-length protein, only 4 antibodies bound strongly enough to Sotatercept for usage with immunoprecipitation followed by SAR-PAGE and Western single blotting. The adapted protocol allows the detection of 0.1 ng/mL Sotatercept in just 50 μL human serum. None of the 3 commercial ACVR2-ELISAs was able to detect Sotatercept and the 2 tested surrogate proteins, even in the μg/mL range. As for Luspatercept, only IPG-IEF/2D-PAGE generated discrete isoforms. Due to the long serum half-life, the SAR-PAGE method will be able to detect Sotatercept for several weeks and will be very useful in doping testing.

AB - A method for the detection of Sotatercept (ACE-011, ACVR2A-Fc) in human serum is presented. The method is a modification of a recently published protocol for Luspatercept (ACE-536, ACVR2B-Fc), another erythropoiesis stimulating fusion protein. Out of 27 tested antibodies against either the extracellular domain of ACVR2A or the full-length protein, only 4 antibodies bound strongly enough to Sotatercept for usage with immunoprecipitation followed by SAR-PAGE and Western single blotting. The adapted protocol allows the detection of 0.1 ng/mL Sotatercept in just 50 μL human serum. None of the 3 commercial ACVR2-ELISAs was able to detect Sotatercept and the 2 tested surrogate proteins, even in the μg/mL range. As for Luspatercept, only IPG-IEF/2D-PAGE generated discrete isoforms. Due to the long serum half-life, the SAR-PAGE method will be able to detect Sotatercept for several weeks and will be very useful in doping testing.

KW - Journal Article

U2 - 10.1002/dta.2346

DO - 10.1002/dta.2346

M3 - Journal articles

C2 - 29193906

SN - 1942-7603

VL - 10

SP - 927

EP - 937

JO - Drug testing and analysis

JF - Drug testing and analysis

IS - 6

ER -

Detection of Sotatercept (ACE-011) in human serum by SAR-PAGE and western single blotting

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