Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein

Gaikwad, Satish S.; Lee, Hyun-Jeong; Kim, Ji-Ye; Choi, Kang-Seuk

A total of 30 chicken sera comprising three groups with each group of 10 were used in the study. These sera were kept at the OIE reference laboratory for Newcastle disease, Animal and Plant Quarantine Agency (APQA), Korea. First group were sera taken from mock-infected SPF chicken (NDV antibody negative). Second and third groups were sera taken 14 dpi from SPF chickens vaccinated with IDE deleted recombinant Newcastle disease virus (rNDV) [4] and NDV LaSota strain [19] , respectively, which kindly supplied by the OIE reference laboratory for ND, Korea. All vaccinated sera were proved to be serologically positive by a HI test using NDV antigen. All sera were available with the laboratory. Microtiter plates were coated with 100 μL of 1 μg/mL rERP protein in 50 mM carbonate/bicarbonate buffer pH 9.6, and incubated overnight at 4°C. Plates were washed three times with PBST and blocked with 150 μL per well PBST with 5% skim milk powder solution at 37°C for 2 hours. The chicken anti-NDV serum (1:400) was added to each. After 1-hour incubation at 37°C, plates were washed three times with PBST. One hundred microliters horseradish peroxidase-conjugated goat anti-chicken IgG (Pierce) at 1:2,000 dilution was added to each well and plates were incubated at 37°C for 1 hour. Then plates were washed three times with PBST and 100 μL TMB (3,3,5,5-tetramethylbenzidine) was added to each well. Tekan, Männedorf, Switzerland).