Tmb substrate

Spike ectodomains were tested for antibody- or ACE2-binding in ELISA assays as described (32 (link)). Assays were run in two formats: antibodies/ACE2-coated or spike-coated. For the first format, the assay was performed on 384-well plates coated at 2 μg/ml overnight at 4°C, washed, blocked, and followed by twofold serially diluted spike protein starting at 25 μg/ml. Binding was detected with polyclonal anti–SARS-CoV-2 spike rabbit serum (developed in our lab), followed by goat anti-rabbit HRP (Abcam, Ab97080) and TMB substrate (Sera Care Life Sciences). Absorbance was read at 450 nm. In the second format, serially diluted spike protein was bound in wells of a 384-well plates, which were previously coated with streptavidin (Thermo Fisher Scientific) at 2 μg/ml and blocked. Proteins were incubated at room temperature for 1 hour, washed, then human mAbs were added at 10 μg/ml. Antibodies were incubated at room temperature for 1 hour, washed, and binding detected with goat anti-human HRP (Jackson ImmunoResearch) and TMB substrate.

Serum concentrations of MxR and 3 × 1 were measured by ELISA. Briefly, Nunc maxsorp 96-well plates (Thermo Fisher, cat#442404) were coated with 100 ng of goat anti-human Fab (Jackson ImmunoResearch, cat#109-005-097) for 90 min at 4 °C. Wells were washed three times with 1×DPBS and then incubated with 1×DPBS containing 1% bovine serum albumin (Sigma-Aldrich, cat#A2153) for 1 h at room temperature. Antigen-coated plates were incubated with serum for 90 min at 4 °C. A standard curve was generated with serial two-fold dilutions of palivizumab. Wells were washed three times with 1×DPBS followed by a one-hour incubation with horseradish peroxidase-conjugated goat anti-human total Ig at a dilution of 1:6000 (Invitrogen, cat#31412). Wells were then washed four times with 1×DPBS followed by a 5–15 min incubation with TMB substrate (SeraCare, cat#5120-0053). Absorbance was measured at 405 nm using a Softmax Pro plate reader (Molecular Devices). The concentration of antibody in each sample was determined by reference to the standard curve and dilution factor.

4 μl of refolded MHCs loaded with photo-cleavable peptides (MHC-J) (0.5 μM) were mixed with 1 μl of the target peptide (50 μM) to be exchanged with, and exposed to 365 nm UV for 60 min on ice. An ELISA assay was performed to quantify the UV-exchange efficiency as described below. Briefly, NeutraAvidin plates (ThermoFisher, 15507) were washed four times with wash buffer (phosphate buffered saline (PBS) containing 0.05% Tween‐20 and 0.1% BSA) and blocked with blocking buffer (PBS containing 2% BSA) for 1 h at 37˚C. Wells were incubated with either 100 μL UV-exchanged samples or MHC-J at 5 nM for 1 h at 37˚C. A folded MHC complex made in house at eight serial two‐fold dilutions, starting from 32 nM were used as positive controls. After washing four times with wash buffer, wells were incubated with HRP‐conjugated β2m antibodies (Rockland, 1:5k dilution) in blocking buffer for 1 h at 37˚C. Wells were washed four times again before incubating with 100 μL TMB substrate (Seracare, 5120‐0047). The TMB reaction was quenched after 5 min using 1 M sulfuric acid. The OD at 450 nm was measured on a Spectramax Plate Reader. UV exchange efficiency was calculated as background-subtracted OD450 of UV-exchanged samples divided by background-subtracted OD450 of the MHC-J sample.

Nunc Immuno MaxiSorp 96-well plates (Thermo Scientific) were coated with SARS-CoV-2 spike protein (Sino Biological) at 1 µg/mL in PBS and incubated overnight at 4 °C. After incubation, plates were washed once with wash buffer (0.05% Tween-20 in 1× PBS), then blocked with casein for 2 to 3 h at 25 °C. Mouse or hamster sera were diluted in casein block buffer starting at 1:25 followed by 3× serial dilutions and incubated in plates for 1 h at 25 °C. Plates were then washed three times and incubated with either rabbit anti-mouse IgG-HRP (Jackson Immuno) or goat anti-hamster IgG(H+L)-HRP (SouthernBiotech) diluted in casein for mouse or hamster samples, respectively. After 1-h incubation at 25 °C, plates were washed three times, then developed using TMB substrate (SeraCare). Development was halted using stop solution (SeraCare). For each sample, ELISA endpoint titer was calculated in GraphPad Prism software, using a four-parameter logistic curve fit to calculate the reciprocal serum dilution that yields an absorbance value (450 nm) of 0.2.