Tmb 3 3 5 5 tetramethylbenzidine

SARS-CoV-2 spike protein-specific immunoglobulin G (IgG) and immunoglobulin A (IgA) titres in serum and bronchoalveolar lavage fluid (BALF) were determined using ELISA. Serums were isolated from whole blood of mice after centrifugation. Bronchoalveolar lavage (BAL) of the lungs of mice was performed with 1 mL of 1× PBS, which yielded approximately 700 μL of BALF. Next, 0. 2 μg of S proteins (Sino Biological, Beijing, China) of the Wuhan-Hu-1, B. 1. 617. 2, and B. 1. 1. 529 strains were coated on a Costar ELISA 96-well plate (Corning, NY, USA) and incubated at 4°C overnight. The plate was subsequently washed with 1× PBST and blocked with 1× PBST containing 1% BSA at 37°C for 1 h. After the plate had been washed six times with 1× PBST, four-fold diluted serum was added to the wells. The plate was washed six times with 1× PBST again and incubated with either horseradish peroxidase-conjugated goat anti-mouse IgA (1:10,000; Abcam, UK) or goat anti-mouse IgG (1:10,000; ZSGB-BIO, China) at 37°C for 1 h. After washing, absorbance values at 450 nm and 630 nm were measured with TMB (3,3’,5,5’-tetramethylbenzidine; Beyotime, China) used as the substrate. IgG and IgA antibody titres in the serum were calculated using GraphPad Prism v9. The end point titres were defined as the highest reciprocal serum dilution, which was 2.1-fold higher compared to the negative control.

Phages derived from the library screening were checked using polyclonal phage ELISA. EGFR fragment (0.2 μg/well) or BSA as a control was used to coat wells of a 96-well plate at 4 °C overnight. After being blocked for 2 h at room temperature, phages from each round of screening were added to appropriate wells. Then, the anti-M13-HRP secondary antibody (Sino Biological, Shanghai, China) was added, and the plates were incubated. TMB (3, 3′, 5, 5′-Tetramethylbenzidine) (Beyotime Institute of Biotechnology, Haimen, China) was added. The reaction was stopped with sulfuric acid after color development. Absorbance of each well was measured at 450 nm by an automated microplate reader (Bio-RAD 680, Bio-RAD, Hercules, CA, USA).

2019-nCoV spike protein-specific IgA and IgG titres were detected with ELISA. Briefly, 0.2 μg 2019-nCoV spike protein (Sino Biological, Beijing, China), and B.1.1.529 spike protein (Sino Biological) were coated overnight onto the Costar ELISA plates (Corning, NY, USA), respectively. The 2019-nCoV spike protein was fused with a polyhistidine tag at the C-terminus, while the B.1.1.529 spike protein was fused with the bacteriophage T4 fibritin and a polyhistidine tag at the C-terminus. After blocking with 0.05% Tween 20-containing PBS and 1% bovine serum albumin at 37°C for 1 h, the plates were rinsed 6 times with 0.05% Tween 20-containing PBS, the diluted sera were added to the wells by 4-fold serial dilutions. After washing 6 times with 0.05% Tween 20-containing PBS, the plates were exposed to horseradish peroxidase-conjugated goat anti-mouse IgA (1:10,000; Abcam, UK) or goat anti-mouse IgG (1:10,000; ZSGB-BIO, China) at 37°C for 1 h. TMB (3,3’,5,5’-tetramethylbenzidine; Beyotime, China) was employed as a substrate to determine the antibody responses by measuring the absorbance at 450 and 630 nm. The end point titres were defined as the highest reciprocal serum dilution, which was 2.1-fold higher compared to the negative control.