Anti chicken igy peroxidase rabbit antibody

The production of specific antibodies against Dg-Cys-his was examined by Western blotting, as described previously [4 (link),5 (link)]. Purified Dg-Cys-his was separated using a 14% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, USA). The membrane was blocked overnight at 4 °C with PBS-T containing 1% skim milk. The membranes were incubated at 25 °C with the isolated plasmas from the immunized chickens, washed three times with PBS-T, and incubated at 25 °C with anti-chicken IgY peroxidase rabbit antibodies (Sigma-Aldrich, A9046) diluted at 5000×. Finally, the membranes were incubated with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) to visualize the peroxidase reaction. To analyze the specificities of antibodies, the His-tagged recombinant protein of D. gallinae copper transporter 1 (Dg-Ctr1-N-his) was also incubated with plasma from the Dg-Cys-his-immunized chickens [4 (link)] in the same manner as described above. To visualize Dg-Ctr1-N-his, SDS-PAGE and CBB staining was also performed as described above.

Antibody titers in plasma samples were determined by ELISA. The purified Dg-Cys-his was coated onto the wells of 96-well plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) (100 ng/well) for 16 h in a carbon-bicarbonate buffer. After washing each well three times with PBS, PBS containing 0.05% Tween 20 (PBS-T) and 1% BSA was added and incubated at 37 °C for 2 h. The wells were then washed five times with PBS-T, and plasma samples diluted at 2000×, 4000× 8000×, and 16,000× with PBS were added. After incubation for 1 h at 25 °C, the wells were washed five times with PBS-T and incubated with anti-chicken IgY peroxidase rabbit antibodies (Sigma-Aldrich, A9046) diluted at 5000× with PBS-T for 1 h at 25 °C. Finally, the wells were washed five times with PBS-T and allowed to react with the TMB one-component substrate (Bethyl Laboratories, Montgomery, TX, USA) for 20 min at 25 °C, in the dark. The reaction was quenched with 0.18 M H₂SO₄, and the absorbance was measured at 450 nm. The assay was performed in duplicate.

Western blotting was performed to confirm the production of specific antibodies against rCP-PD PRM, rCP-PD TFM, and rCP-PD NFM, and to analyze the cross-reactivity of immunized plasma. rCP-PD proteins were separated via 13% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck). Membranes were blocked with 0.05% Tween 20 in phosphate-buffered saline (PBST) containing 3% skim milk at 4°C overnight. Membranes were then incubated with isolated immune plasma (1:1000) at 25°C for 1 h, washed with PBST three times, and incubated with anti-chicken IgY peroxidase rabbit antibody (Sigma-Aldrich, St. Louis, MO, USA) at 25°C for 1 h and washed with PBST three times. Finally, the peroxidase signal was visualized by incubating the membrane with Immobilon Western Chemiluminescent Horseradish Peroxidase (HRP) Substrate (Merck) for 5 min at 25°C.