96 well half area elisa plates

Unless otherwise stated, 96-well half-area ELISA plates (Corning Incorporated) were coated overnight at 4–8°C with 50 μl/well of 20 μg/ml solutions of different proteins in PBS. The next day the plates were washed twice with 0.05% Tween 20 in PBS (Tween/PBS). All subsequent steps were performed at room temperature. Between addition of consecutive reagents plates were washed 4 times with Tween/PBS. All recombinant receptors were obtained from R&D Systems. The enzymatic reaction was performed with the use of TMB Substrate Reagent Set (BD Biosciences) as the HRP substrate.

The binding antibody titers using ELISA were assessed according to methods previously published [20 (link)]. Briefly, 96-well half-area ELISA plates (Corning (Tewksbury, MA, USA)) were coated with the recombinant Washington strain (WA-1) spike protein (expressed as a prefusion-stabilized S-2P construct) [21 (link)] at 2 μg/mL in PBS, followed by overnight incubation at 4 °C. The next day, the plates were washed three times with PBS-T (PBS containing 0.05% Tween 20) and blocked with 5% milk (Oxoid skim milk powder, Thermo Scientific, Waltham, MA, USA) in PBS (w/v) for 1 h at room temperature. Serially diluted plasma samples in duplicate were added to the plates and incubated for 2 h at room temperature. The plates were washed three times, and a goat anti-monkey IgG–horseradish peroxidase secondary antibody (Nordic-MUBio (Susteren, the Netherlands)) was added for 1 h at room temperature. The plates were washed three times again, and 1-Step Ultra TMB-ELISA substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added for 5 min. The reaction was stopped with 1 M H₂SO₄, and the absorbance was measured at 450 nm with background correction at 570 nm. The data were analyzed using Prism v9.4.1 using a 4-parameter logistic curve fit.

To test IgG antibody avidity against RBD protein, threefold serial dilutions of 1/20 diluted mice sera, were added to ELISA plates (96 well half-area ELISA plates; Costar, Corning, Catalog # 3690) coated over night at 4°C with 1μg/ml RBD. After incubation at RT for 1h, the plates were washed once in PBS-0.01% Tween, and then washed 3x with 7M urea in PBS-0.05%Tween or with PBS-0.05% Tween for 5min every time. After washing with PBS-0.05%Tween, goat anti-mouse IgG conjugated to Horseradish Peroxidase (HRP) (Jackson ImmunoResearch, West Grove, Pennsylvania) was added 1/1000 and incubated for 1h at RT. Plates were developed and read at OD₄₅₀ nm.