Elisa diluent

Serum samples were diluted 1:10 with ELISA diluent (BioLegend. Cat. 408804, San Diego, CA, USA). IgG antibodies were removed from the diluted serum samples. For this purpose, we took advantage of the SureBeads™ Magnetic Beads system (BIO-RAD. Cat. 161-4823). The system contains magnetic beads designed to capture IgG or IgA antibodies. The beats can attach IgG or IgA antibodies from the FC region. The procedure was carried as follows: 100 μL of magnetic beads (SureBeads protein G) were transferred to 1.5 mL tubes. The beads were washed three times with 1 mL of PBS + 0.01% Tween 20. After magnetization of the beads, the wash solution was discarded and 350 μL of diluted serum samples were added into the tube. Samples were left rotating for 10 min at RT and magnetized again. The supernatants (serum samples depleted in IgG) were collected and stored at −80 °C until analysis.

The gene-transfected DO-11.10 cells were lysed using RIPA buffer (50 mM Tris–HCL pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail; Nacalai Tesque) on ice for one hour. Cell lysates were centrifuged at 10,000 × g for ten minutes at 4 °C and the supernatants were used for ELISA. The stock silk solutions (10 mg/mL in 2 M LiBr) were diluted with 1 mM Tris–HCl (pH 8.0) to a concentration of 0.2 mg/mL. One hundred microliters of the cell lysate, culture supernatants from hybridoma cells, and diluted silk solutions were applied to 96 well plates and incubated overnight at 4 °C. After washing thrice with PBS, each well was blocked using ELISA Diluent (BioLegend, San Diego, CA, USA) at room temperature for one hour. After five washes with PBS and Tween 20, formalin-inactivated As (1.8 × 10⁸ CFU/mL) was diluted, applied to the wells, and incubated at room temperature for 90 min. Binding was detected using sequential incubation of plates with anti-As-4A MAb and HRP-conjugated anti-mouse IgG Fc (abcam, ab97265), followed by incubation with ELISA POD Substrate TMB solution (Nacalai Tesque). After color development, the reaction was stopped with 2N H₂SO_4, and the absorbance was read at 450 nm using a microplate reader (iMark™ Microplate Reader; Bio-Rad).

Binding antibody units (BAU/mL) against the receptor binding domain (RBD) of the SARS-CoV-2 Spike from the Wuhan strain amongst the groups were measured as previously described (36 (link)). Briefly, 96 well plates were coated with recombinant RBD of SARS-CoV-2 WT spike overnight at 4°C and subsequently blocked with ELISA Diluent (Biolegend) for 1 h at 37 °C. Serum samples were serially diluted, including a negative serum control, anti-S antibody as positive control (Dianova, CSB-RA332450A0GMY), and a calibrator. Pre-diluted samples and controls were incubated on the coated plate for 1 h at 37 °C. After washing, HRP conjugated secondary antibody (goat anti human IgG Fc gamma fragment specific, Dianova) was added and incubated for 1 h at 37 °C. After washing, the plate was tapped dry and incubated with substrate (1 Step Ultra TMB, Pierce) for 5-10 min at RT in the dark until the positive control showed distinct blue staining. The reaction was stopped with 2 M H₂SO₄ and absorbance was measured at 450 nm. Normalization was performed by: (sample-negative control)/(calibrator-negative control). The sample dilution was used to calculate sample BAU/mL by fitting hyperbolic curves in GraphPad Prism using the correction factor of the WHO Standard 20/136 measurements, which is defined as 1000 BAU/mL.