SARS-CoV-2 Spike Protein RBD ELISA

ELISAs were completed using plates coated with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein using a previously described protocol with slight modifications. (6 , 25 (link)) Plasmids for expressing this protein were provided by Florian Krammer (Mt. Sinai). SARS-CoV-2 RBD proteins were produced in 293F cells and purified using nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen). The supernatant was incubated for 2 hours with Ni-NTA resin at room temperature before the Ni-NTA resin was collected using gravity flow columns and the protein was eluted. After buffer exchange into phosphate-buffered saline (PBS), the purified protein was stored in aliquots at -80°C. ELISA plates (Immulon 4 HBX, Thermo Scientific) were coated overnight at 4°C with 50 μL per well of PBS or a 2 μg/mL recombinant protein diluted in PBS. The next day, ELISA plates were washed 3 times with PBS containing 0.1% Tween-20 (PBS-T) and blocked for 1 hour with PBS-T supplemented with 3% non-fat milk powder. Prior to testing in ELISA, serum samples were heat-inactivated at 56°C for 1 hour. Serum samples were serially diluted in 2-fold in 96-well round-bottom plates in PBS-T supplemented with 1% non-fat milk powder (dilution buffer), starting at a 1:50 dilution. Next, ELISA plates were washed 3 times with PBS-T and 50 μL serum dilution was added to each well. Plates were incubated for 2 hours at room temperature using a plate mixer. Plates were washed again 3 times with PBS-T before 50 μL of horseradish peroxidase (HRP) labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories) (1:5,000) or goat anti-human IgM-HRP (SouthernBiotech) (1:1,000) secondary antibodies were added. After 1 hour incubation at room temperature using a plate mixer, plates were washed 3 times with PBS-T and 50 μL SureBlue 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL) was added to each well. Five minutes later, 25 μL of 250 mM hydrochloric acid was added to each well to stop the reaction. Plates were read at an optical density (OD) of 450 nm using the SpectraMax 190 microplate reader (Molecular Devices). Background OD values from the plates coated with PBS were subtracted from the OD values from plates coated with recombinant protein. A dilution series of the IgG monoclonal antibody CR3022, which is reactive to the SARS-CoV-2 spike protein, was included on each plate as a control to adjust for inter assay variability. The IgG CR3022 monoclonal antibody was included on both IgG and IgM plates, and an anti-human IgG-HRP secondary antibody was added to these standardization wells on both IgG and IgM plates. In essence, the CR3022 monoclonal antibody was used to set the OD threshold on each plate and to ensure that the same OD threshold was used on all plates, including both IgG and IgM assays. Serum antibody concentrations were reported as arbitrary units relative to the CR3022 monoclonal antibody. Plasmids to express the CR3022 monoclonal antibody were provided by Ian Wilson (Scripps). All samples were first tested in duplicate at a 1:50 serum dilution. Samples with an IgG and/or IgM concentration above the lower limit of detection (0.20 arbitrary units) were repeated in at least a 7-point dilution series to obtain quantitative results.