Dynabeads m 270 carboxylic acid

100 μL of approximately 30 mg/mL carboxylic acid coated magnetic beads (Dynabeads M-270 Carboxylic Acid, Invitrogen) were transferred to a 1.5 mL microcentrifuge tube and washed twice with 100 μL of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 5, Sigma-Aldrich) for 10 min. The beads were then activated with 50 μL of freshly prepared 50 mg/mL N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Sigma-Aldrich) and 50 μL of 50 mg/mL N-hydroxysuccinimide (NHS, Sigma-Aldrich) with rotation at room temperature for 30 min. The activated beads were washed twice with 100 μL of 25 mM MES buffer (pH 5) and then mixed with 60 μg of anti-CEA capture antibody (10-C10F, Fitzgerald) or anti-EGFR capture antibody (DYC1854–2, R&D systems), respectively. After overnight incubation with rotation at 4 °C, the beads were magnetically collected to remove supernatant and blocked by 100 μL of 50 mM ethanolamine (Sigma-Aldrich) in phosphate-buffered saline (PBS, Corning Cellgro) for 60 min at room temperature with rotation. Finally the capture antibody labelled magnetic beads were washed four times with 100 μL of washing buffer (0.1% BSA and 0.1% Tween-20 in PBS), resuspended in 200 μL PBS, and stored at 4 °C.