Goat anti chicken igy secondary antibody conjugated with hrp

The persistence kinetics of IgY-R when intranasally delivered were evaluated at the local level in the oropharyngeal region and at the systemic level in serum. For this, six hamsters were inoculated with a dose of 10 µg of IgY-R per gram of body weight (1X), intranasally, and four hamsters with PBS for the control group. Subsequently, oropharyngeal swab and blood samples from gingival vein were taken at 24 and 48 h after the administration of IgY-R and processed by soaking swabs into 100 µL of PBS, allowing blood to coagulate at room temperature for 1 h, then centrifuging at 5000 g for 5 min to obtain serum. The solution obtained from swabs and serum were diluted 1:2 and 1:50 with 1% (w/v) of skim milk, respectively, and subjected to ELISA assay similar to that described above, incubating samples at 37°C in a plate previously fixed with RBD protein and blocked with skim milk, prior to incubation with Goat anti-Chicken IgY secondary antibody conjugated with HRP (Genscript) diluted 1:20000, at 37°C for 1 h for the subsequent TMB addition and reading at 405 nm.

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).

To perform the assay, a fixative solution containing 1 µg/mL of SARS-CoV-2 RBD protein (GenScript) was prepared in carbonate-bicarbonate buffer (pH 9.6) and then a Nunc MaxiSorp flat bottom plate (Sigma) was coated with 100 µL of the fixative solution and incubated at 4°C overnight. The next day, the plate was washed five times with DPBS 0.05% (v/v) Tween-20 buffer (0.05% DPBS-T) and blocked with 3% (w/v) of skim milk (BD Biosciences) in 0.05% DPBS-T for 2 hours at room temperature, the plate was then washed five times with 0.05% DPBS-T. 100 µL of sera diluted 1/2000 (week 1 post-vaccination to week 7 post-vaccination) and 100 µL of purified total IgY antibodies (0.3 mg/mL) diluted 1/800 (week 1 post-vaccination to week 10 post-vaccination) both with 1% (w/v) of skim milk were added to the plate and incubated for 1 hour at 37°C. Later, wells were washed five times with 0.05% DPBS-T and immediately incubated with 100 µL of Goat anti-Chicken IgY secondary antibody conjugated with HRP (Genscript) diluted 1/2000 in 1% non-fat milk in 0.05% DPBS-T for 1 hour at 37°C. The plate was then washed five times with 0.05% DPBS-T and incubated with 100 µL of 3,3’,5,5’-tetramethylbenzidine (TMB) for 15 min at room temperature. The reaction was stopped by the addition of 50 µL of 2N H₂SO₄ per well, and the plate was read at 450 nm using an Epoch 2 microplate reader (Bioteck).