Janus automated liquid handling workstation

Serial 3.16 fold dilutions of serum were prepared with assay diluent [1% ECL^™ Blocking Agent (GE Healthcare Biosciences, Piscataway, NJ) in 1X PBST (PBS—0.1% Tween 20)], starting with 1:10 dilution or 1:100 dilution and higher. For each sample, a minimum of 3 dilutions were tested. Serial dilution was performed by Janus® automated liquid handling workstation (PerkinElmer, Waltham, MA). All other steps were performed manually. Plates were blocked for 1 h with 5% ECL^™ Blocking Agent in 1X PBST at room temperature (24 °C ± 2), 150 μl per well, on a lab rotator set at 650 rpm. All incubations for subsequent steps were at 37 °C for 1 h with shaking at 650 rpm. After removal of the blocking agent, 25 μl of sample per well was added and the plate incubated. After each incubation plates were washed 4 times with 150 μl per well of 1X PBST using an automated plate washer (ELx405VRS, Biotek, Winooski, VT). 25 μl of biotin-labeled mouse anti-human IgG (Fc specific) (Biotrend Chemicals LLC, Destin, FL) at 1 μg/ml in assay diluent, followed by 25 μl of Streptavidin-Sulfo Tag^™(MSD) (1:500 dilution in assay diluent) was added to each well in subsequent steps. 150 μl of 1X Read Buffer T (MSD) was added to each well and the plate was immediately read on the Sector Imager 6000 (MSD) (read time: 70 s/plate). RLU for each spot was exported to Microsoft Excel.