Ccd camera

Purified RBD was loaded at 0.2 μg/well and electrophoretically separated by SDS-PAGE under non-reducing conditions and transferred to nitrocellulose membranes using an e-blot device (GenScript Laboratories, USA). The membranes were blocked with 5% (w/v) non-fat milk in PBS with 0.1% of Tween 20 at pH 7.4 and incubated overnight at RT. Then, membranes were washed three times for 5 minutes each with Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T) and incubated for two hours at RT with serum of a hamster immunized with a Newcastle disease virus expressing the S1 sub-unit of SARS-CoV-2 [22 (link)] (1:250) in 5% non-fat milk. After three washes with TBS-T, anti-Hamster IgG antibody conjugated to HRP (Abcam, USA) was added to the membrane at 1:5000 dilution in 5% non-fat milk and incubated for two hours at RT. Finally, the membranes were washed three times with TBST-0.1%, incubated with luminol (Azure Biosystems, USA) as a substrate and revealed with a CCD camera (Azure Biosystems, USA).

Bacterial lysate, pellet and culture supernatant samples were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane using an e-blot device (GenScript Laboratories). The membranes were blocked with Azure Chemi Blot clocking buffer (Azure Biosystems, Dublin, CA, USA) with shaking for 1 h at room temperature. Membranes were washed three times for 10 min with phosphate-buffered saline (PBS)-Tween 20 and were incubated with purified monoclonal antibodies (0.4 μg/mL). Membranes were washed three times with phosphate-buffered saline-Tween 20. The immune reaction was performed with peroxidase-labelled goat IgG anti-mouse antibody (GenScript Laboratories) using radiance as a substrate and revealed by a CCD camera (Azure Biosystems). For bacterial lysates and pellets, 10-fold dilutions were prepared with the previously quantified samples (500, 50, 5, 0.5, 0.05 and 0.005 μg/mL). For bacterial culture supernatants, 2-fold dilutions were prepared with the supernatant (1:2, 1:4, 1:8 and 1:16) in PBS at pH 7.0. For recombinant TBDT protein, 500 ng was used. For molecular weight estimation of the studied proteins, we used a pre-stained molecular weight marker that ranged from 26,600 to 180,000 Da (Sigma Aldrich Co.) and a molecular weight marker that ranged from 30,000 to 120,000 Da (GenScript Laboratories).

To demonstrate the recognition activity of the viral RBD protein by the IgY-R pool, a Western Blot assay was carried out starting with an SDS-PAGE run of SARS-CoV-2 RBD protein (GenScript) at a rate of 0.3 µg per well, and the WB-MASTER Protein Standard (Genscript) as ladder, following the same procedure as described above. The content of the resulting gel was transferred to a nitrocellulose membrane using the eBlot L1 Protein Transfer System, according to the manufacturer’s recommendations. Then, the membrane was subjected to a 10-min wash with TBST wash buffer (tris-buffered saline and 0.1% Tween 20), and blocking was performed with PBS buffer supplemented with 0.1% Tween 20 and 3% milk, for 1 h. Subsequently, a wash step was performed, and the membrane was incubated with a dilution of IgY-R antibodies (1mg/mL) at a ratio of 2:5000 in the Azure Protein Free Blocking Buffer for 2 h. Then, another wash step was applied, and Goat anti-Chicken IgY secondary antibody conjugated with HRP (Genscript) at a ratio of 2:5000 in the Azure Protein Free Blocking Buffer was added, following incubation for 2 h, and a wash step prior to incubation with luminol (Azure Biosystems) for 2 min. Afterward, the membranes were revealed and photographed in a CCD camera (Azure Biosystems).