Live SARS-CoV-2 virus nAb assay
±, infection
INFECTION, SECONDARY
Antibody-mediated neutralization of live wild-type SARS-CoV-2 (WA isolate, passage 3, Vero-E6 cells) was measured at Battelle using a microneutralization assay (The SARS-CoV-2 stock was produced by infecting Vero-E6 cells [African green monkey kidney, passage 31; originally obtained from BEI Resources (catalog no. NR-596)] with CDC-provided material (2019-nCoV/USA-WA1/2020; GenBank, accession number MN985325.1; passage 3) at a multiplicity of infection of 0.001 in Eagle’s minimum essential medium supplemented with antibiotics and 2% fetal bovine serum. Virus-containing supernatant was harvested after 72 hours of incubation at 37° ± 2°C and 5 ± 2% COFor the ELISA, plates were incubated with anti-nucleocapsid protein primary antibody cocktail (clones HM1056 and HM1057) (EastCoast Bio; catalog nos. HM1056 and HM1057) for 60 min at 37°C. The plates were washed, the secondary antibody [goat anti-mouse IgG horseradish peroxidase conjugate, Fitzgerald; catalog no. 43C-CB1569) was added to the wells, and the plates were incubated for 60 min at 37°C. Refer to U.S. patent application nos. 17/447,022 and 17/336,443 for further details. The optical density value of each sample well was measured with a microplate reader using a wavelength of 405 nm and a 490-nm reference. Each sample was tested independently in singlet by one operator on one test plate following the standard operator procedures. The same sample was then tested by a second operator in singlet on a different plate on the same day. If necessary, repeat testing of any samples was performed in singlet by one operator on a different test day. The final reportable value for each sample was the median MN