Tracking SARS-CoV-2 Antibody Responses

SARS-CoV-2 spike protein-binding IgG, IgM, and IgA titers were determined by ELISA using remnant sera obtained from baseline (D –1) and 1, 3, and 7 days after patients received CCP (D1, D3, and D7, respectively). Briefly, microtiter plates (Costar, Corning) were coated with 25 μL of 2 μg/mL purified spike protein (32 (link), 55 (link), 75 (link)) in phosphate-buffered saline (PBS) overnight at 4°C, washed with 1× PBS/0.1% Tween (PBS-T), blocked with 3% (v/v) milk (Bio-Rad)/PBS-T for 1 hour at room temperature (RT), washed, and incubated with heat-inactivated sera for 2 hours at RT. Plates were then washed, incubated with isotype-specific HRP-labeled goat anti-human IgG (Thermo Fisher Scientific 31410), IgM (MilliporeSigma A6907), or IgA (MilliporeSigma A0295) for 1 hour at RT. Following final washes, plates were incubated with ultra-TMB ELISA substrate (Thermo Fisher Scientific), and color development was stopped by addition of 0.5 M sulfuric acid (MilliporeSigma). Well absorbances at 450 nm (A₄₅₀) were determined using a Cytation 5 (BioTek). The endpoint titer was determined as the highest dilution to give a signal 3 times the background A₄₅₀ (wells with no sera).