Viruses and clinical samples
Serotype 1, 5, 6, 7, 9 and 10 fowl adenoviruses (FAdV-1, FAdV-5, FAdV-6, FAdV-7, FAdV-9 and FAdV-10) were from ATCC. Serotype 8 fowl adenovirus (FAdV-8), Marek’s disease virus (MDV), avian reticuloendotheliosis virus (REV), Infectious bronchitis virus (IBV), chicken infectious anemia virus (CAV), H9N2 avian influenza virus (AIV) and subgroup J avian leukosis virus (ALV) were kept in our laboratory. The Serotype 4 fowl adenovirus FAdV-4 strains SD15, SDWF15, AHFY15, JSCZ15 and JH13 were isolated as previously described . Thirty two liver samples of chickens were from a diseased chicken flock naturally infected with FAdV-4. All the studies associated with the viruses were performed in cabinet under ABSL2 facility.
Cells and plasmids
The LMH cell (the chicken liver cell line, from ATCC) was cultured in F12/DMEM (Gibco, NY, USA) with 10% FBS (Lonsera, Shanghai, China). The plasmids pcDNA3.1-F2 and pcDNA3.1-F1 kept in our laboratory could efficiently express the fiber-2 and fiber-1 protein of FAdV-4 respectively.
Antibodies and protein
Chicken sera specific to different serotypes of FAdV were provided by Sinopharm Yangzhou VAC Biological Engineering Co., Ltd. The secondary antibodies including FITC-labelled goat anti-mouse IgG and HRP-labelled rabbit anti-chicken IgY were from Sigma (CA, USA). The purified fusion protein GST-fiber1-S (about 40kD) with the shaft domain of the fiber-1 protein of FAdV-4 (corresponding to the amino acids 79–208 of the fiber-1 protein) was generated in our laboratory.
Generation of mAbs
The 6-week-old BALB/C mice were immunized with the purified GST-fiber1-S fusion protein four times every 10 days as previously described . At day 3 post the last immunization, the spleen cells from one immunized mouse were collected and fused with SP2/0 cells as previously described . The hybridoma cells secreting antibodies specific to the fiber-1 of FAdV-4 were screened using IFA. In the IFA analysis, the LMH cells infected with FAdV-4 were used as antigen. After the subcloning of the positive hybridoma cells, the mAbs secreted by these positive clones were further identified by IFA, western blot and immunoprecipitation. The ascites of these identified mAbs were prepared and purified as previously described . All the mice studies were performed under ABSL2 facility. At the end of the experiment, all the mice were first anesthetized with isofluorane and then euthanized by cervical dislocation.
Indirect immunofluorescence assay (IFA)
LMH cells transfected with pcDNA3.1-F1 and pcDNA3.1-F2 respectively or infected with FAdV-4 for three days were fixed with the fixed solution (acetone and ethanol 3:2) for 5 min, and then the IFA was performed according to the previous report .
Immunoprecipitation and immunoblotting
LMH cells transfected with pcDNA3.1-F1 or infected with FAdV-4 for 3 days were lysed in lysis buffer (CST, MA, USA) with protease and phosphatase inhibitors cocktail (CST), and PMSF (Beyotime, Shanghai China). The lysates were collected after centrifugation for 20 min at 12000 rpm and the supernatants were used for western blot and immunoprecipitation as previously described .
The ELISA plates were coated with the capture mAb (100 μL/well) diluted in 0.1 M carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C. And the coated ELISA plates were then blocked with PBST (pH 7.4, containing 0.05% Tween-20) with 1% FBS and 5% skimmed milk for 2 h at 37 °C. After the ELISA plates were washed once with PBST, the diluted samples (100 μL/well) in PBST were subsequently added and incubated for 1 h at 37 °C. After the ELISA plates were washed with PBST for four times and the mAb conjugated with HRP (100 μL/well) diluted with 1% skimmed milk in PBST was added to each well. After incubation for 1 h at 37 °C and washing another four times with PBST, the TMB substrate solution (100 μL/well) was added into each well, and the ELISA plates were incubated for 15 min at 37 °C in the dark. 2 M H2SO4 (50 μL/well) was then added to stop the reaction and the value of OD450 was determined by an ELISA plate reader. The condition for the ELISA was optimized by analysis of the OD450 value and the positive/negative ratio (P/N) of the samples at different conditions. The cut-off value of the ELISA was evaluated by using 60 FAdV-4 free samples including tissue samples and chicken cloacal swab samples, and was determined by calculating the two folds of the arithmetic mean of these FAdV-4 free samples. For detection of the clinical tissue samples, the liver samples from a diseased chicken flock were homogenated (1 g tissue in 1 mL PBS), and the supernatants from the homogenates were diluted in 1:10 with PBS for the ELISA test.