Anti sars cov 2 nucleocapsid

Monoclonal antibodies and their corresponding isotype controls used were the following: Alexa Fluor 594 anti-NOS2/iNOS (1 µg/test; BioLegend #696804), Alexa Fluor 594 rat IgG2bκ isotype (1 µg/test; BioLegend #400661), rat anti-mouse/human iNOS CXNFT (10 µg/mL; eBioscience #14-5920-82) (37 (link)), purified rat IgG2aκ isotype (10 µg/mL; BD Biosciences #553927), anti-SARS-CoV-2 Nucleocapsid (5.6 µg/mL; Sino Biological #40143-MM05), and IgG1κ isotype from murine myeloma (5.6 µg/mL; Sigma-Aldrich #M9269). Conjugated secondary antibodies used (1:1000) were the following: goat anti-mouse IgG1 Alexa Fluor 488 (Invitrogen #A-21121), goat anti-mouse IgG1 Alexa Fluor 568 (Invitrogen #A-21124), goat anti-rat IgG Alexa Fluor 568 (Invitrogen #A-11077), and goat anti-rat IgG Alexa Fluor 647 (Invitrogen #A-21247).

VN antibodies present in the sera were detected as described previously (22 (link), 23 (link)). In short, inactivated serum samples (at 56°C for 30 min) were first diluted 1:10 followed by two-fold serial dilutions. Vero cells (ATCC CCL-81) were seeded in a 96-well tissue culture plate. Diluted sera were mixed with 200 TCID₅₀ of SARS-CoV-2 Wuhan-Hu-1 and incubated for 1 h at 37°C. Serum–virus mix was added to a monolayer of Vero cells and further incubated at 37°C, 5% CO₂. After 8 h of incubation, cells were fixed using 4% Paraformaldehyde (PFA) and incubated for 30 min at room temperature. Next, PFA was removed, and cells were incubated for 15 min with 80% methanol. For staining, plates were blocked using 1% BSA in PBS–0.05% Tween 20 for 30 min at 37°C. SARS-CoV-2 was detected using a 1:1,000 dilution of rabbit polyclonal anti–SARS-CoV-2 nucleocapsid (Sino Biological). After 1 h of incubation at 37°C, cells were washed with PBS–0.05% Tween 20 and incubated with a 1:1m000 dilution of anti–rabbit–IgG–Alexa Flour 488 (Invitrogen). Finally, cells were washed twice with PBS–0.05% Tween 20. Fluorescent cells were counted using the C.T.L. S6 Ultimate-V Analyzer, and data were analyzed using CTL ImmunoSpot^® software. Neutralizing antibody titers are expressed as the dilution that gave a 50% reduction of stained cells (NT50).