N h2so4

To determine the tetanus toxoid and SARS-CoV-2 RBD-specific antibody titres, 96-well plates were coated overnight with 0.5 µg/ml of either tetanus toxoid (AJ vaccines) or SARS-CoV-2 (2019-nCoV) Spike RBD-His recombinant protein (Sino biological, Cat. 40592-V08B-100). Coated plates were washed, blocked for 1 h with PBS 5% BSA and incubated overnight at 4 °C with serial dilutions of sera. Specific IgA antibodies were detected using anti-human IgA-Biotin (Southern Biotech, Cat. 2050-08) followed by streptavidin-HRP (Invitrogen, Cat. N100) and specific IgG antibodies were detected using anti-human IgG-HRP (Southern Biotech, Cat. 2040-05). Detection antibody incubation was performed at room temperature for 1 h. After washing 5 times with PBS-T, Tetramethylbenzidine (TMB) Substrate (Invitrogen, Cat. 88-7324-88) was added. The reaction was stopped by the addition of 2 N H2SO4 (Sigma-Aldrich: Cat. 84736). Optical densities were measured on Spectramax (Molecular devices). Optical densities were measured on Spectramax plus 384(Molecular devices). OD values were further plotted against respective sample dilutions, and areas under the curve (AUC) were quantified using Graphpad Prism 9.3.1.

Plates were coated overnight with 0.4 µg/well of either N, RBD, S recombinant proteins, or alternatively 10⁴ PFU/well of UV-inactivated SARS-CoV-2, and blocked for 2 h with PBS containing 2% bovine serum albumin (PBS-2% BSA) at 37 °C. Serum was serially diluted, and the bronchoalveolar lavage (BALF) samples were tested at 1:1 dilution in PBS-2% BSA and incubated for 1 h at 37 °C, and then incubated with anti-human IgG-HRP antibody (Fapon), anti-hamster IgG-HRP or anti-mouse total IgG, IgG1, IgG2c conjugated with streptavidin-HRP (Southern Biotech), all diluted 1:5000. After 5 washes, plates were revealed with 1-Step ultra TMB substrate solution (Biolegend) for 15 minutes in the dark, and reaction was stopped by adding 2 N H₂SO₄ (Sigma). Plates were read in 450 nm and results were expressed as raw optical density (OD). The antibody titer was determined by the sera dilution that yielded 50% of the maximum antibody reactivity to the antigen in the ELISA.