Plasma biomarker measurements

For measurements of plasma levels of eNAMPT, IL-6, and TNF-α, a meso-scale ELISA platform was utilized (Meso Scale Diagnostics, Rockville, MD) as we have described.^5,10 Each biotinylated antibody, specific for each analyte, was diluted in the coating buffer to a concentration of 10 µg/ml. A volume of 200 µl of diluted antibody was next mixed with 300 µl of a different linker for each analyte and vortexed. After 30 min RT incubation, the reaction was stopped with 200 µl of free biotin solution. Then, 600 µl of this 10× U-PLEX linked biotinylated antibody solution was added to a tube and mixed. The volume was supplemented with Stop and 50 µl of coating solution added to each well in 96 well plate and incubated for 1 h at 800 r/min shaking at room temperature. After washing three times, the plate was supplemented with 25 µl of diluent and 25 µl of calibrator or samples/standards to each well. The plate was incubated for 1 h at 800 r/min at RT. After washing three times with TBS-T, the plate was supplemented with 50 µl/well of 1× detection antibody solution to each well before being again incubated for 1 h at 800 r/min shaking at RT. After washing three times with TBS-T the plate was supplemented with 150 µl/well of 2 × Read Buffer T. The plate was finally detected in the imager and the absolute concentration values were calculated based on standards. ¹⁰