Alp conjugated streptavidin

For competitive ELISA used in epitope mapping of mAbs, 2 μg/mL recombinant RBD-his (Sino Biological, Beijing, China) was added in 384-well plates and incubated at 4 °C overnight. 50 μg/mL mAbs per well were added. The plates were incubated at 37 °C for 1 h and then washed. Biotinylation of mAbs (the top 20 neutralizing Abs and 81A11, previously reported SARS-CoV CR3022^{24 (link)}) were performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) according to the manufacturer’s protocol and purified using MINI Dialysis Unit (ThermoFisher, 69576). 500 ng/mL biotinylated mAbs were added to each well, and the plates were incubated at 37 °C for 1 h. ALP-conjugated streptavidin (Mabtech, Sweden, 3310-10) was added at 1:1000, followed by an incubation of 30 mins at 37 °C. For the quantification of bound IgG, PNPP (Thermo Fisher) was added at 1 mg/mL and the absorbance at 405 nm was measured by the MultiSkan GO fluoro-microplate reader (Thermo Fisher).

PVDV membrane plates (Millipore, MA, USA) were coated with anti-human IFNγ antibody (clone: 1-DK1, Mabtech Inc, #3420-3-1000, conc.: 2 μg/ml) overnight. Expanded and overnight rested PBMC samples (see above) were counted and 3 × 10⁵ PBMCs were added per well with S or N overlapping peptide pools (both Miltenyi, Germany) at 1.25 μg/ml peptide, and incubated overnight. Medium alone was used as a negative control. Pools of 23 MHC-I restricted peptides from human Cytomegalovirus, Eppstein Barr virus and Influenza virus (CEF, Anaspec Inc.) and 35 MHC-II restricted peptides from human Cytomegalovirus, Epstein Barr virus, Influenza virus, Tetanus toxin, and Adenovirus 5 (CEFTA, Mabtech Inc.) were used as positive controls. IFNγ secretion was detected with a biotinylated anti-human IFNγ antibody (clone: 7-B6-1, Mabtech Inc, #3420-6-1000, conc. 0.5 μg/ml) and ALP conjugated-Streptavidin (Mabtech Inc). Spots were developed with 1-Step BCIP/NBT-plus reagent (Mabtech Inc.) for 20 min. Membranes were dried and spots were analyzed and counted on an ImmunoSpot CTL analyzer. A response was considered positive only if there were ≥50 SFCs/10⁶ PBMC after subtracting the value of the matched negative control.