Visulize factor 7 antigen elisa kit

Cell culture medium from cells with transient or stable expression of rFVII mutants was harvested and the cells were washed briefly with ice-cold phosphate-buffered saline (PBS) (Lonza) before lysis in RIPA buffer (Sigma-Aldrich) supplemented with 1x Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Cell lysates and cell culture medium were pre-cleared by centrifugation and total protein content in the lysates was measured using the BCA protein assay kit (Thermo Fisher Scientific). ELISA was performed on cell culture medium and lysates using the VisuLize™ Factor VII Antigen ELISA Kit (Affinity Biologicals, Ancaster, ON, Canada) according to the manufacturer's protocol.
Colorimetric output was measured in a SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). FVII levels for the transiently transfected Huh7 cells were normalized by subtracting the endogenous FVII levels.