Tmb substrate

ELISA was used to determine anti-OVA or anti-SARS-CoV-2 spike IgG titers. Briefly, 96-well microtiter plates (Thermo Fisher) were coated with 2.0 µg/mL of OVA (Invivogen) or SARS-CoV-2 spike protein (RayBiotech) overnight at 4°C. Plates were washed with 0.05% Tween-20 in PBS (PBST) and blocked with 1% BSA/PBST. Mouse serum samples were two-fold serially diluted in PBST, added to the blocked plates, and incubated at 37 °C for 1 h. Following incubation, plates were washed with PBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 (Southern Biotech, cat#1070-05, 1:5000) or goat anti-mouse IgG2c (Southern Biotech, cat# PA1-29288, 1:4000) for 1 h. Plates were washed with PBST and TMB substrate (BD Bioscience) was added. Reactions were stopped with 50 µl 2 N H₂SO₄. Plates were read at OD 450 nm with a SpectraMax Plus plate reader (Molecular Devices). The antibody titer is defined as the dilution in which absorbance is more than 2.1 times of the blank wells.

Surrogate virus neutralization assay, which allows assessment of virus inhibition without the BSL-3 facility requirement, was performed as described previously [25 (link)]. HRP-conjugated RBD was provided by Optipharm (Cheongju, Republic of Korea), which was performed using the HRP conjugation kit (Abcam, Cambridge, UK). SARS-CoV-2 human angiotensin-converting enzyme 2 (hACE2) receptor protein was purchased from GenScript (Piscataway, NJ, USA). After coating the plates with 100 ng of hACE2 using carbonate coating buffer in 96-well plates overnight at 4 °C, plates were washed with PBST and subsequently blocked with BD OptEIA assay diluent (BD Bioscience, San Diego, CA, USA) for 1 h at RT. Sera collected from mice 1 week after the final immunization were serially diluted in PBS and incubated at 56 °C for 30 min for complement inactivation. After heat inactivation, equal volumes of inactivated sera and HRP-conjugated RBD were mixed and inoculated into each well to be incubated for 1 h at 37 °C. Wells were washed with PBST and chromogenic reactions were developed using TMB substrate (BD Bioscience, San Diego, CA, USA). Reactions were stopped with 2N H₂SO₄ and absorbance values at 450 nm were measured. Percentage of inhibition was calculated as follows: Inhibition (%) = (1 − sample OD₄₅₀/control OD₄₅₀) × 100.

Costar 3590 96-well ELISA plates were coated for16h at 4 °C with 100 µL of PS (Sigma) at 20 μg/ml in 200-proof Molecular Biology ethanol or calf thymus DNA (Sigma) at 10 μg/ml in PBS. DNA plates were allowed to evaporate at RT after coating. Plates were washed 5 times with 200 µL of PBS and then blocked for 1 h with 200 µL of PBS 3% BSA. Plasma from patients was diluted at 1:100 in PBS 1% BSA and incubated for 2 h at 37 °C. Plates were washed again five times and incubated with anti-human IgG-HRP (GE Healthcare) for 1 h at 37 °C. Plates were washed five more times and TMB substrate (BD Biosciences) was added until the desired color was obtained. The reaction was stopped by with Stop buffer (Biolegend) and absorbance was read at 450 nm. Positive and negative controls were the same used for XO determinations. The mean OD at 450 nm from triplicate wells was compared with the same dilution of the reference positive serum to calculate relative units (RU). Anti-PS and anti-DNA levels were determined in all available samples at one-month follow-up (n = 443).

Sera was collected from mice 14 days post-primary and 14 days post-secondary injection and diluted according to the expected antibody response (between 1:10 and 1:5000). Plates were coated with 100 μl of the full-length M72 protein at 1 μg/mL. Following washing (PBS plus tween 20) and blocking (SuperBlock, Scytek Laboratories), plates were incubated with diluted serum for 1 h followed by anti-mouse IgG, IgG1, or IgG2a-HRP secondary antibody (Bethyl Laboratories) and TMB substrate (BD). Plates were read at 450 nm. Antibody titers were determined by calculating titer of each sample at OD 0.3.

The specificity of the immune sera (diluted from 1.500 to 1:20,000) was tested in an ELISA. Two micrograms of BotI-like enriched fraction and BSA (irrelevant antigen) were coated overnight at 4°C in wells of a Maxisorp plate. After the plates were washed five times with phosphate-buffered saline (PBS)–Tween 20 (0.05%) (PBS-T), the residual protein-binding sites in the wells were blocked by PBS, 5% milk. Then, 100 µl of serially diluted immune and non-immune sera was added to separate wells and incubated for 1 h at room temperature (RT). The wells were washed five times with PBS-T [PBS plus 0.1% (v/v) Tween-20] and incubated with an anti-LAMA phosphatase alkaline-conjugated antibody (Sigma, St. Louis, MO, USA) (1:8,000). The reaction was developed using TMB substrate (catalog number 555214, BD Biosciences, San Diego, CA, USA) for 15 min at room temperature and stopped with 100 µl/well of 2 N H₂SO₄. After incubation for 20 min at room temperature, titers were estimated by measuring the optical density at 450 nm with a plate reader (Thermo Electron Corporation Multiscan EX, MA, USA). All experiments were performed in duplicate, and the mean values and standard deviations were calculated.

ELISA plates were coated with 400 ng/well purified TSG MAb in carbonate–bicarbonate buffer (pH 9.6) (Sigma, St. Louis, MO, USA, C3041) and held at 37 °C for 1 h. TSG was used to capture VLPs (or mutated VLPs) for antibody characterization because its binding epitope was excluded from the neutralizing sites. The plates were washed three times with PBST (PBS with 0.05% Tween-20, pH 7.3) followed by blocking with 0.5% skim milk (Sigma, 70166) in PBS for 30 min. After the removal of the blocking buffer, 1 µL of cell lysates containing VLPs (or mutated VLPs) was added at a dilution of 1:100 and incubated at 37 °C for 1 h. After washing three times, the conjugates (100 ng/well) were incubated for 1 h. After the final washing, freshly prepared TMB substrate (BD biosciences, Franklin Lakes, NJ, USA, 555214) was added and allowed to stand for 15 min. The reaction was stopped with 1 N HCl and measured at 450 nm with a Sunrise^TM Absorbance Reader (TECAN, Männedorf, Switzerland). Each test was performed in triplicate.