Detection of erythropoiesis stimulating agents in urine samples using a capillary Western system

Abstract

The presence of erythropoiesis stimulating agents (ESAs) in the urine samples collected from athletes is detected using traditional Western blotting following either size-based separation (SDS/SAR-PAGE) or isoelectric focusing (IEF). Although there is an important testing effort, there is little doubt that ESAs are still abused in sports and that reducing the costs of the tests might increase the number of tests and improve deterrence. The capillary electrophoresis system developed by Protein Simple may be useful to this end. This platform is fully automated and could be easily implemented in anti-doping laboratories, which would contribute to the improvement of the overall assay performance and standardization of the method. Such an automated system could be of interest during major sports events, such as the Olympic Games, where a high number of samples needs to be analyzed in a short period of time. From the experiments conducted so far, we conclude that the technique is promising, with the sensitivity and reproducibility needed to screen ESAs in human urine samples.

Detection of activin receptor type IIA and IIB‐Fc fusion proteins by automated capillary immunoassay

“…Automated capillary Western (Simple Western) was performed using the Wes system (Protein Simple, San Jose, CA, USA) and adapted from previous study 25 . The analyses were performed with the 12‐ to 230‐kDa Separation Module (Protein Simple, SM‐W004) according to the manufacturer's instructions with some modifications brought to the instrument default settings.…”

miR‐375 induces adipogenesis through targeting Erk1 in pancreatic duct cells under the influence of sodium palmitate

“…Calculations were performed using Compass Software (version 3.1.8). The peak area of target proteins were normalized to the peak area of β‐actin for cytoplasmic proteins or total protein peak area for secretion samples to calculate arbitrary units (AU; Desharnais et al, 2018).…”

“…Calculations were performed using Compass Software (version 3.1.8). The peak area of target proteins were normalized to the peak area of β-actin for cytoplasmic proteins or total protein peak area for secretion samples to calculate arbitrary units (AU;Desharnais et al, 2018).2.10 | Transfection20 nM miR-375 inhibitor was applied to ARIP duct cells for 48 h with lipofectamine. Transfection efficiency was analyzed by qRT-PCR(Jayamohan et al, 2019).2.11 | StatisticsEach experiment was repeated at least three times and the data were given as mean ± SEM.…”

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…Most analytical assays enabling the detection of erythropoietin (EPO)‐receptor agonists in doping control blood or urine samples utilize gel electrophoretic approaches, optimized to accommodate the diverse physico‐chemical properties of recombinant human erythropoietin, its analogs and/or EPO‐mimetic agents . The established methods have been shown to provide robust detection windows unaffected by hyperhydration, yet faster and less laborious ITP alternatives have been desirable and the applicability of an integrated capillary electrophoresis (CE)/Western blotting system was assessed by Desharnais et al Following established protocols, 15 mL of urine are concentrated by ultrafiltration and, subsequently, ESAs are immunopurified before loading into autosampler plates for automated CE‐supported analysis. The CE system was equipped with a 12–230 kDa nano‐capillary (5 cm × 100 μm) based separation module, which allowed for immobilizing proteins to the capillary wall after electrophoresis followed by incubation with primary and secondary antibodies for chemiluminescent detection.…”