Anti-HER2 monoclonal antibodies m4D5 (provided by Genentech) and biotinylated 6E2 [16 (link)] were used as the capture and detection antibody, respectively. HER2 ECD standard was originally purchased from Oncogene Sciences. For ELISA, the capture antibody (m4D5, 5 μg/ml, 50 μL) was coated in PBS to a 96-well plate for overnight incubation at 4°C. After wash with PBST, the plate was blocked with 5% BSA for 1 h at room temperature (22°C). HER2 ECD standard and serum samples were diluted in the MBB buffer [13 (link)] and incubated for 1-h at 22°C. Diluted biotinylated detection antibody (50 μL, 1 μg/mL, with MBB) was added to each well for a 1 hr-incubation at room temperature. Streptavidin-conjugated horseradish peroxidase (HRP) (R&D systems) was used as the secondary antibody to detect the antigen-antibody complex. The plate was washed three times with PBST (0.1% Tween 20 in PBS) in-between incubations. Following six washes with PBST to remove excess detection antibodies, 50 μL of Tetramethyl Benzidine (TMB) substrate (0.1 mg/mL, 0.05 M phosphate-citrate buffer, pH 5.0) was incubated in each well at 22°C. The reaction was stopped within 15–30 min with 50 μL of 2 M H2SO4, and the data was collected at 450 nm (absorbance filter) using the SpectraFluor reader (Tecan).