Tween 20 pbs solution

Blood samples were collected upon study enrollment using a vacuum-containing system. Plasma levels of sST2, GDF-15, H-FABP, IGF-BP2, and suPAR were measured by using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, USA). Preparation of reagents and measurements were performed according to the manufacturer’s instructions. In brief, patient samples and standard protein were added to the wells of the ELISA plates (Nunc MaxiSorp flat-bottom 96 well plates, VWR International GmbH, Austria) and incubated for two hours. Plates were then washed using a Tween 20/PBS solution (Sigma Aldrich, USA). Then, a biotin-labelled antibody was added and incubated for another two hours. Plates were washed another time, and streptavidin–horseradish-peroxidase solution was added to the wells. After adding tetramethylbenzidine (TMB; Sigma Aldrich, USA) a color reaction was generated. Values of optical density (OD) were determined at 450 nm on an ELISA plate-reader (iMark Microplate Absorbance Reader, Bio-Rad Laboratories, Austria).

Blood samples were obtained on the day of hospitalization and thus one day before the TAVR procedure using a vacuum-containing system. The collection tubes were centrifuged, and the plasma was separated from the blood components and then frozen at −80 °C. Samples were measured at equal time points under similar conditions.
Plasma levels of sST2, GDF-15, H-FABP, IGF-BP2 and suPAR were analyzed by using enzyme-linked immunosorbent assay (ELISA) kits (GDF-15: DY957, H-FABP: DY1678, IGF-BP2: DY674, suPAR: DY807, R&D Systems, Minneapolis, MN, USA). Manufactures’ instructions were performed for the adequate preparation of reagents. Therefore, serum samples and standard proteins were placed into the wells of ELISA plates (Nunc MaxiSorp flat-bottom 96-well plates, VWR International GmbH, Vienna, Austria) and incubated for two hours. The plates were treated with Tween 20/PBS solution (Sigma Aldrich, St. Louis, MO, USA). Afterwards, a biotin-labeled antibody was added and incubated for another two hours. A washing process was performed, and streptavidin–horseradish–peroxidase solution was added to the wells. A color reaction was generated after adding tetramethylbenzidine (TMB; Sigma Aldrich, USA). Optical density was determined at 450 nm on an ELISA plate reader (iMark Microplate Absorbance Reader, Bio-Rad Laboratories, Vienna, Austria).