Streptavidin ap

BALB/C mice were supplied by The Jackson Laboratory, USA; Harlan, Italy; Charles River, Germany, Italy. Animal experiments were approved by the Cantonal Autorithies of Cantone Ticino. Groups of 6- to 8-week-old female BALB/c mice were injected intravenously with 100 μg of purified mAbs or 2 mg of total IgG purified from PA96 patient. After 16 h, 2 μg of human GM-CSF were injected. Serum samples were collected on day 1 and day 5. GM-CSF was quantified with a sandwich ELISA. Briefly, 10 μg ml⁻¹ of an antibody that bound to site II of GM-CSF was used to coat 96-well Maxisorp plates (Nunc), which were then blocked with PBS+10% FBS (Gibco). All sera and GM-CSF, which was used as standard (range 3.4–600,000 pg ml⁻¹), were titrated and tested in parallel under different conditions: in one plate all samples were supplemented with 25% (vol/vol) of an alkaline dissociation buffer (2.5% Triton X-100, 2 M ethanolamine, 0.15 M NaCl, pH 11.6), in the other plate all samples were supplemented with 25% (vol/vol) of PBS+10% FBS. Plates were left overnight at RT. Detection of captured GM-CSF was made with 1 μg ml⁻¹ of a biotinylated antibody that bound to site I of GM-CSF for 1 h at RT, followed by binding of 0.5 μg ml⁻¹ streptavidin-AP (Jackson ImmunoResearch) for 1 h at RT. Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm.

MBC culture supernatants were diluted in PBS and mixed with SARS-CoV-2 Wu RBD mouse Fc-tagged antigen (Sino Biological, 40592-V05H) or with biotinylated BQ.1.1 or XBB.1 RBDs (Acrobiosystems) at a final concentration of 20 ng ml⁻¹ and incubated for 30 min at 37 °C. The mix was added for 30 min to ELISA 384-well plates (NUNC, P6366-1CS) pre-coated overnight at 4 °C with 4 µg ml⁻¹ human ACE2 (produced in house) in PBS. Plates were washed with PBS containing 0.05% Tween-20 (PBS-T), and RBD binding was revealed using secondary goat anti-mouse IgG-AP (Southern Biotech, 1032-04) or Streptavidin-AP (Jackson ImmunoResearch). After washing, pNPP substrate (Sigma-Aldrich, 71768-25G) was added and plates were read at 405 nm after 1 h.