Antibody response to HER2-ICD protein detected by ELISA. High binding 96-well microtiter plates were coated with HER2-ICD protein (10 μg/ml, 200 μl/well) for overnight at 4°C. After washing, and blocking with 1% BSA in PBS for 2 hours, serially diluted patients' sera (200 μl/well) were added to the wells in triplicate and incubated overnight at 4°C. Control serum from a normal donor was used as a negative control. Alkaline phosphatase conjugated anti-human IgG was added to the plate after washing, and color was developed using p-nitrophenyl phosphate. Absorbance at 405 nm was measured and the differences from the control serum at each dilution was plotted. The data represents the mean absorbance for each serum dilution ± standard deviation for each patient depicted.