Cellular Fibronectin-EDA ELISA Protocol

Cellular Fn-EDA levels in the plasma were measured by sandwich enzyme-linked immunosorbent assay (ELISA) as described.(16 (link)) Briefly, microtiter plates were coated overnight at 4°C with primary antibody for cellular Fn-EDA (IST-9, 10 μg/mL, Abcam) diluted in 50 mM sodium carbonate buffer. 50 μl of plasma samples (diluted 1:2 in PBS) were incubated for 2 h in the coated wells at RT. After five washes biotinylated secondary antibody to FN (2 μg/ml diluted in blocking buffer) was added to wells and incubated for 1 h at RT. Following five washes avidin HRP solution (1:1000) in blocking buffer was added to wells and incubated for 30 minutes. Microtiter plates were washed five times, before adding 3, 3′, 5, 5′-tetramethylbenzidine substrate solution (Sigma) to the wells and the colorimetric reaction was stopped with H₂SO₄ (2M) after 10 min. Results were read in an ELISA microplate reader at A₄₅₀ nm. Human cellular Fn (Sigma) was used for standards.