The washing procedure was applied for three times (ELISA Washer ELX 50, Bio-tek Inst.). After the washing step, 100 μL of substrate/chromogen solution were added to each well and mixed gently and incubated for 30 min at room temperature (20–25°C) in the dark. Finally, 100 μL of the stop solution (1 M H2SO4) were added to each well and the absorbance was measured at 450 nm in ELISA plate reader (ELX 800, Bio-tek Inst.)