Goat anti chicken igy

Total chicken and penguin IgY were purified using the Pierce™ Chicken IgY Purification Kit kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Purified chicken or penguin IgY were loaded on a 4-15% Mini-protean TGX stain-free gel (Bio-Rad, Hercules, USA) and separated by SDS-electrophoresis 1 hour at 120V by duplicated. One of the gels was used for protein staining with Coomassie blue. The other gel was used for protein transference onto Trans-Blot Turbo Transfer Packs membrane (Bio-Rad, Hercules, USA). The membrane was blocked 2 hours with 1% HSA (Sigma-Aldrich, St. Louis, MO, USA)/PBS at RT, washed three times with PBST, and incubated at 4°C overnight with HRP-conjugated goat anti-turkey IgY (Mybiosource, San Diego, CA, USA), or goat anti-chicken IgY (Sigma-Aldrich, St. Louis, MO, USA) antibodies dilution 1:400 in PBS. Immunoreactive proteins were visualized with TMB solution (Promega, Madison, WI, USA).

To measure total plasma IgM and IgY EIA plates (Corning) were coated with 2 μg/mL goat‐anti‐chicken‐IgM (Bethyl Laboratories) or 0.8 μg/mL goat‐anti‐chicken‐IgY (Sigma Aldrich) overnight at 4°C. Plates were blocked with PBS/3% skim milk and incubated with chicken plasma. Bound IgM or IgY was detected with goat‐anti‐chicken‐IgM‐HRP (Bethyl Laboratories) or goat‐anti‐chicken‐IgY‐HRP (Sigma Aldrich) and developed with TMB substrate (Thermo Fisher Scientific). OD was measured at 450 nm. To measure antigen‐specific IgM and IgY, plates were coated with 10 μg/mL KLH or BSA. For detection of the Cμ‐CH1 domain plates were coated with 2 μg/mL ms‐anti‐chicken‐ Cμ‐CH1 (M1) (Southern Biotech). All subsequent steps were performed as for detection of total plasma IgM and IgY.