Anti human il 6

IL-6 levels were measured by sandwich ELISA. Briefly, a 96-well
microtiter plate (Corning™ Clear Polystyrene flat-bottomed, 96-Well,
medium binding), was coated with 100 μL of capture antibody (4
μg/mL, anti-human IL-6, BioLegend) diluted in PBS and incubated overnight
at 4°C. After blocking with 1% BSA in PBS for 2 hours at room
temperature, samples were added and incubated overnight at 4°C. For
detection, plates were incubated with biotinylated detection antibody (1
μg/mL in blocking buffer, Biotin anti-human IL-6) for 2 hours at room
temperature, followed by addition of HRP-streptavidin (BioLegend) for an
additional 2 hours at room temperature. The reaction was visualized by the
addition of TMB. The reaction was stopped by addition of 2N sulfuric acid and
the absorbance was measured at 450 nm by plate reader. Wells were washed
thoroughly three times in PBS-Tween between every step. CRP was analyzed using
ELISA according to the manufacturer’s protocol (Enzo Life Sciences Inc.
Farmingdale, NY, USA).