Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue

Abstract

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.

Selected routine laboratory tests in the clinical assessment of patients with obstructive sleep apnea

“…According to Cummins et al, the haemoglobin and haematocrit levels were significantly higher among patients with severe OSA compared to those without OSA, although in every group, values stayed within the normal range [19]. Moreover, the haemoglobin and haematocrit levels were negatively correlated with mean SpO2 as a result of hypoxia, which stimulates the production of erythropoietin and consequent increased erythrocytes production [20].…”

Annual banned‐substance review 16th edition—Analytical approaches in human sports drug testing 2022/2023

“…Within the class S.2 of WADA's Prohibited List, ERAs represented 70% of all reported AAFs in 2021, 126 suggesting that ERAs still play an important role as illicitly administered drugs in sport, despite the risks purportedly and evidently associated with their non‐therapeutic use 133 . Test methods regarding ERAs have been continuously optimized and complemented concerning enhanced anti‐doping applications and additional strategies to determine the presence of a natural variant of EPO (c.577del) and in the light of medicinal research 134 and pharmaceuticals potentially influencing the EPO biosynthesis in humans 135–137 To verify the presence of a single nucleotide polymorphism (SNP) in the c.577del EPO gene, which results in a frameshift and, consequently, in an increased molecular mass of the corresponding EPO protein, Yi et al presented a testing protocol a employing clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9‐based testing approach 70 . With designated primers, the relevant SNP‐modified EPO sequence is polymerase chain reaction (PCR)‐amplified from 3 μL of blood and the amplicons and incubated with a complex formed from dCas9 and a variant‐specific single‐guide DNA.…”

Tubular Endogenous Erythropoietin Protects Renal Function against Ischemic Reperfusion Injury

“…Epo pretreatment examined the effects of exogenous Epo on renal function. We have invented the deglycosylation-coupled Western blotting method for Epo in blood, urine, and tissue [28][29][30]. Our method improved the sensitivity and specificity of Epo detection, especially in tissue.…”