Tween 20

Fifty microliter of commercial 250 nm Ni–NTA-coated MNPs (Nanomag‐D, Micromod, 4.9 × 10¹¹ particles mL⁻¹) were washed twice with 500 µL of 0.1 M PB Tween 20 (Scharlab) (0.05%, v v⁻¹) by removing the supernatant after trapping the particles with a magnetic concentrator (Dynal‐Biotech). Then, 500 µL of the recombinant RBP Gp17, expressed and purified as described by Costa et al. [20 (link)], at a final concentration of 5 µM was added to the MNPs and incubated in an orbital shaker at 500 rpm, 20 °C for 2 h. After MNP functionalization, the above-mentioned washing procedure was performed to remove the supernatant containing the unbound protein. In the next step, 500 µL of a 5% (w v⁻¹) bovine serum albumin (BSA) (Sigma-Aldrich) solution in 0.1 M PB was added to the MNPs to block the remaining free surface and incubated at the same conditions as during functionalization for 1 h. The supernatant was discarded and the MNP-RBPs conjugates were washed with 0.1 M PB Tween 20 (Scharlab) (0.05% v v⁻¹), resuspended in 50 µL 0.1 M PB, and kept at 4 °C as stock. For comparison purposes, 50 µL of commercial 250 nm streptavidin-coated MNPs (Nanomag‐D, Micromod, 4.9 × 10¹¹ MNPS mL⁻¹) were functionalized with 500 µL of a biotinylated E. coli polyclonal antibody (LS-C56164, LSBio) at a final concentration of 50 μg mL⁻¹, following the same procedure described above.

Serum from the rabbit of different groups were analyzed by enzyme‐linked immunosorbent assay (ELISA) to determine sera antibody titers. ELISA plate (Corning, USA) was coated with 1 µg/ml SARS-CoV-2 Spike S1 + S2 ECD-His recombinant protein (Sino Biological, China) in Dulbecco’s phosphate-buffered saline (DPBS) (ThermoFisher, USA) for 2 h at room temperature. Plate was washed for 3 times with DPBS + 0.05% Tween 20 (Scharlau, Spain) and then blocked with PBS + 1% BSA (ThermoFisher, USA) + 0.050% Tween-20 for 2 h at 37 °C. The plate was washed for 3 times then incubated with rabbit sera and SARS-CoV-2 Spike antibody (Sino Biological, China) for 2 h at 37 °C. After washing for 3 times, the plate was incubated with HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (ThermoFisher, USA) for 50 min at room temperature. Final washing was done for 3 times and then developed for colorimetric reaction with Pierce TMB substrate (ThermoFisher, USA) for 10 min. The reaction was stopped with 1 N HCl and the plate was read at 450 nm wavelength within 30 min.