Horseradish peroxidase conjugated goat anti chicken igy antibody

Groups were set as previously mentioned. Serum of preimmunization, 7 d after initial immunization, and 7 d after second immunization was obtained after centrifugation at 3,000 rpm for 5 min at 4°C. Serum from 5 chickens per group was used to determine the EtTA4-specific IgY antibody by indirect ELISA (Huang et al., 2015 (link)). Briefly, 96-well microtiter plates (Costar) were coated with rEtTA4 (50 μg/mL) overnight at 4°C. After washing 3 times with 0.01 M PBS containing 0.05% Tween-20, the plates were blocked with 5% skim milk powder in PBS containing 0.05% Tween-20 for 2 h at 37°C, and 100 μL of the serum samples diluted 1:50 in PBS containing 0.05% Tween-20 and 5% skim milk powder was added for 1 h at 37°C. After washing, the plates were incubated with 1:40,000 dilution of horseradish peroxidase-conjugated goat-anti-chicken IgY antibody (Abcam) for 1 h at 37°C. Tetramethylbenzidine (Sigma) substrate was used to develop colors and the optical density at 450 nm was measured on a microplate reader (Thermo Scientific, United States). All serum samples were determined with 3 repeats.

Poly (D, L-lactide-co-glycolide) (molar ratio of D, L-lactide to glycolide, 65:35, MW = 40,000–75,000) and polyinyl alcohol (MW=31,000–50,000, 98–99% hydrolyzed) were purchased from Sigma-Aldrich (St. Louis, MO). The Ni-NTA column was the product of GE Healthcare Life Sciences. The Micro BCA Protein Assay Kit was purchased from Thermo Scientific (Rockford, IL). Horseradish peroxidase-conjugated goat-anti-chicken IgY antibody was purchased from Abcam (Cambridge, UK). Mouse anti-chicken CD3-FITC antibody, mouse anti-chicken CD8α-PE antibody, and mouse anti-chicken CD4-PE were the products of Southern Biotechnology Associates (Birmingham, AL). The Toxin Eraser Endotoxin Removal Kit was the product of GeneScript (Piscataway).