Amicon ultra 0.5 3k centrifugal filter devices

After PSA release from ACT, total serum PSA was isolated using the Access Hybritech PSA Assay Kit (Beckman Coulter, Brea, CA, USA). Briefly, a suspension of 100 μl of paramagnetic particles coated with mouse monoclonal anti-PSA (Access Hybritech PSA Assay, Beckman Coulter, Brea, CA, USA) was washed with 500 μl of incubation buffer (50 mM Tris, 150 mM NaCl, pH 7.4, 0.1% Tween-20, 1% BSA) using magnetic separation. The beads were then incubated with 1 ml of neutralized treated serum (containing 750 μl of original serum) for 1h at room temperature (rt) with shaking. Next, the beads were washed with 500 μl of washing buffer (50 mM Tris, 150 mM NaCl, pH 7.4, 1% Triton X-100) using magnetic separation. The immunoadsorbed PSA was eluted in three steps using 100 μl of Gentle Ag/Ab Elution Buffer, pH 6.6 (Thermo Scientific, Rockford, IL, USA) for 30min each. The final elution volume was of 300 μl. Samples were desalted and concentrated to a final volume of 40 μl using Amicon Ultra-0.5 3K Centrifugal Filter Devices (Millipore, Cork, IRL), which had been previously pre-treated with 5% Brij-35 (Sigma-Aldrich, Germany) and used for sample desalting according to the manufacturer's instructions.