2.4. Selenium Measurement and SELENOP Quantification by ELISA and Western Blot Analysis

Plasma Se concentrations were quantified by total reflection X-ray fluorescence (TXRF), as described previously [11,17]. SELENOP concentrations were measured by a validated commercial SELENOP-specific ELISA (selenOtest^TM, selenOmed GmbH, Berlin, Germany) as described [28]. For Western blot (WB) analysis, standard curves were generated using diluted NIST 1950 reference plasma (National Institute of Standards and Technology, Gaithersburg, MD 20899, USA) with known SELENOP values [28,29]. A commercial mouse monoclonal antibody (Selenoprotein P, B-9, Catalog # sc-376858, dilution; 1:500) and an in-house antibody (dilution 1:2000 [28]) were used for SELENOP detection, in combination with horseradish peroxidase-conjugated secondary antibody (Dako rabbit anti-mouse, 1:3000). Samples (0.1 µL of NIST 1950 plasma or patient sample) were separated by 12% SDS-PAGE (Bio-Rad, Stockholm, Sweden), and transferred to a 0.20 µm pore-sized PVDF membrane (Bio-Rad). Membranes were incubated with primary antibodies diluted in 5% milk overnight at 4 °C, washed three times with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20), incubated with secondary antibody diluted in 5% milk for 1 h at room temperature (RT), and developed with enhanced chemiluminescence (WesternBright Sirius HRP substrate, Advansta, San Jose, CA, USA). Digital images were acquired using LiCor Odyssey Fc imaging system, and quantification of immunoreactive bands was carried out by Image Studio Lite (version 5.2).