Sars cov 2 s protein trimer

ELISA micro plates were coated with 2 µg/mL SARS-CoV-2 S-protein trimer (ACRObiosystems) and incubated overnight at 4°C. Microplates were washed 3x with phosphate-buffered saline (PBS) and blocked with 200 µL/well of Protein-Free Blocking Buffer PBST (Cat. #786-665, G-Biosciences) for 2 h at RT.
Mouse bone marrow derived B cells were isolated from treated mice and 1x10⁵ cells were seeded into each well in 200 µL AIMV media and incubated at 37°C for 48 h.
At the end of the incubation period, the cells were removed from each well and each microplate was washed 5x with 200 µL/well of 0.05% Tween 20 in PBST. The samples were then incubated in 100 µL/well of 1:5000 mouse IgG-HRP in PBST for 2 h at RT in the dark before washing 3x in 250 µL PBST. The presence of spike specific IgG was detected by adding 100 µL/well of TMB Substrate System and allowed to incubate for 10 min, by which time a color solution formed, and no longer than 20 min. Enzyme reaction was stopped by adding 50 µL/well of 2N H2SO4 Stop Solution. The samples were analyzed using a CLARIOstar microplate reader (BMG LABTECH) at OD_450nm with OD_540nm as the reference wavelength and analyzed using the MARS software (BMG LABTECH).