Rabbit anti human igg hrp

Ninety-six-well plates were coated with 100 µL per well of 136.3 ng of purified RBD in 50 mM carbonate/bicarbonate buffer (pH 9.6) and incubated overnight (ON) at 4 °C. After blocking 1 h at 37 °C with 5% (w/v) non-fat milk in PBS, plates were incubated with 1:40 diluted serum or plasma sample for 1 h at 37 °C. At the same time, the following controls were tested in each run: (1) a blank reactive control (BRC); (2) a negative sample control (prepandemic serum); and (3) an antigen-free control (without RBD) and a 1:40 positive control during the sample incubation step (named control A). Then, plates were incubated with a 1:3225 diluted rabbit anti-human IgG-HRP (Santa Cruz Biotechnology) for 1 h at 37 °C. Finally, plates were incubated for 13 min with substrate solution (0.5 mg/mL o-phenylenediamine, 0.12% (v/v) H₂O₂ in 50 mM phosphate citrate buffer). Absorbance was measured at 492 nm with a microtiter plate reader (LabSystems Multiskan, Thermo Fisher Scientific). Between every step, plates were washed six times with PBS, 0.05% (v/v) Tween-20 (PBS-T). Dilutions of tested samples and antibodies were prepared in PBS-T containing 0.2% (w/v) non-fat milk.
The same procedure was applied for the iELISA employing stressed RBD sample treated with 100 mM DTT as coating antigen.