Costar elisa plate

SARS-CoV-2 spike protein and spike receptor-binding domain (RBD)-specific IgG and IgA titres were determined using ELISA. Briefly, Costar ELISA plates (Corning, Inc., Corning, NY, USA) were coated overnight with 0.2 μg SARS-CoV-2 spike protein or recombinant RBD protein (Sino Biological, Beijing, China). The plate was blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.05% Tween 20 for 1 h at 37 °C. After washing the plates six times with PBS containing 0.05% Tween 20, sera were added to the wells at 4-fold serial dilutions. Plates were washed six times with PBS containing 0.05% Tween 20 and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (ZSGB-BIO, Beijing, China, 1:10,000) or horseradish peroxidase-conjugated goat anti-mouse IgA (Abcam, Cambridge, UK, 1:10,000) for 1 h at 37 °C. After washing, 3,3’,5,5'-tetramethylbenzidine (TMB) (Beyotime, Shanghai, China) was used as the substrate to detect the antibody responses at 450 and 630 nm. The endpoint of the serum antibody titre was calculated as the reciprocal of the highest dilution, which was 2.1-fold higher than the optical absorbance value of the negative control.

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.

mAb-3F8 was diluted in panning solution (BSA 1% (w/v), dried skim milk 1% (w/v), PBS) and coated on two wells (1.5 μg/well) of a Costar ELISA plate (Corning, Wiesbaden, Germany). Two additional wells were coated with panning solution only; the plate was incubated overnight at 4°C. The L. monocytogenes ATCC 7644 library (≈1 × 10¹⁰ CFU/mL) was diluted in panning solution and added to the panning solution well for pre-incubation for 30 min at RT, while the wells containing mAb-3F8 were blocked with the same solution. The library was further transferred to the wells containing the antibody and incubated 1.5 h at RT. Then, the wells were washed and eluted with 10 μg/mL of trypsin diluted in PBS. The eluted phages were used to infect E. coli TG1 (OD₆₀₀ ≈ 0.5), which was further infected with helperphage M13K07 (MOI 1:20). Infected E. coli TG1 cells were grown overnight at 30°C, 500 rpm. On the next day, the cultures were centrifuged (3,220 ×g, 10 min, RT) and the supernatant containing phage used instead of the library. In total, three panning rounds were done, and the phage eluted after the 2^nd and 3^rd rounds were used to infect E. coli XL1-Blue MRF’, which were diluted, plated on 2xYT-GA agar plates, and grown overnight at 37°C.

Eight‐ to twelve‐week‐old CD137^−/− on the BALB/c background and wild‐type (WT) BALB/c mice were administered 5% Dextran Sodium Sulfate (DSS, MW 36,000–50,000, MP Biomedicals, Santa Ana, CA) in drinking water ad libitum for 7 days; noncolitis control mice were given tap water. At day 7, mice were fasted and 5% DSS was replaced with tap water. At day 8, intestinal epithelial cell integrity was assessed: mice were gavaged with 60 mg/100 g of 30 mg/mL fluorescein isothiocyanate (FITC)‐dextran (4 kDa, Sigma‐Aldrich, St. Louis, MO) in 1XPBS. Blood was collected retro‐orbitally 4 h after gavage, and plasma was collected by centrifugation at 8000 g for 10 min at 4°C. Plasma was diluted with equal amount of 1XPBS. 100 μL of diluted plasma was added in duplicate to a clear 96‐well plate (Costar‐ELISA plate, Corning, Tewksbury, MA). Fluorescence was detected at excitation wavelength 360 with emission wavelength 485 nm on Molecular Devices SpectraMax M2e plate reader. The concentration of fluorescein was determined using serially diluted samples of the tracer in 1XPBS as a standard. Fluorescence images of mice given FITC‐dextran were acquired using an iBox Explorer imaging microscope (UVP). All mouse studies were done according to institutional IACUC protocol approval and NIH guidelines.