Costar 96 well eia ria plates

The overnight coating of Costar 96 Well EIA/RIA plates (Corning) was carried out at 4 °C with 100 µL of 4 µg/mL RBD (RayBiotech, or BSA (used as control) in Coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.8). After 4 washes with 300 µL of PBS, the plate was blocked with 200 µl of a 2% (w/v) ECL Advance Blocking Reagent (GE Healthcare) solution in PBS-T (PBS supplemented with 0.1% (v/v) Tween-20) for 2 h at RT. The plate was washed 4 times with PBS, and then, starting at 2 µg of the purified antibody per well (100 µl), 1:5 serial dilutions in blocking solution were incubated for 1 h 30 min at RT. After 4 washing steps with PBS-T (PBS buffer supplemented with 0.05% Tween-20), 1:2000 HRP-labelled rabbit anti-human IgG (Sigma-Aldrich, #A8792) in blocking solution was added. After 1 h, the plate was washed with PBS and the substrate o-phenylenediamine dihydrochloride SIGMAFAST™ OPD tablet (Sigma-Aldrich, #P9187) was added (following manufacturer's instructions). Reactions were stopped with 50 µL 3 M HCl per well and absorbance was measured at 492 nm. The endpoint titer was determined as the last concentration of each purified antibody showing an absorbance value higher than the value defined as cutoff (mean blank + 3SD). Blank is defined as the values from each ELISA test against BSA (Zrein et al., 1986; Armbruster and Pry, 2008) .