Quantifying SARS-CoV-2 Nucleocapsid Antibodies

The levels of antibodies against SARS-CoV-2 nucleocapsid proteins were quantified as previously described [20 (link), 21 (link)]. SARS-COV-2 nucleocapsid protein was coupled to Luminex beads, following an established protocol (hal-01299922 [22 ] courtesy of National Research Institute for Sustainable Development, France, https://en.ird.fr/). A 25 µL solution containing coupled beads was aliquoted into each well of a 96-well plate. The supernatant was removed while beads were immobilized using a magnetic holder and 1/100 diluted blood plasma was then added to each well. The plate was incubated for 2 h at 25 °C on a plate shaker at 400 rpm, then washed three times with wash buffer. Anti-human lgG secondary antibody (4 µg/mL) was added to the plate and incubated for 30 min on a Corning^® LSE™ digital microplate shaker at 400 rpm. This was followed by three washes, the addition of 1 µg/mL streptavidin–phycoerythrin (Invitrogen) and incubation for 10 min on a plate shaker at 400 rpm. Finally, the plates were read on a Luminex MAGPIX system (Luminex Corporation, Austin, TX, USA) using xPONENT™ software (V.4.3.229).