Horseradish peroxidase hrp conjugated anti mouse igg secondary antibody

Total protein was extracted from 10 females in RIPA buffer (150 mM NaCl, 1.0% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) plus protease inhibitors (Roche Applied Science). Samples were denatured for 5 min at 100°C, electrophoresed on 8% SDS-PAGE gels, transferred onto nitrocellulose membranes (Roche Applied Science) and immunodetected following standard procedures. Membranes were incubated with primary anti-GFP antibody (1 h, 1:1000, Roche Applied Science) followed by horseradish peroxidase (HRP)-conjugated anti-mouse-IgG secondary antibody (1 h, 1:3000, Sigma-Aldrich). Loading control was anti-tubulin (overnight, 1:5000, Sigma-Aldrich) followed by incubation with HRP-conjugated anti-mouse-IgG secondary antibody (1 h, 1:3000, Sigma-Aldrich). Bands were detected using ECL Western Blotting Substrate (Pierce). Images were obtained using the camera system ImageQuant LAS 4000 (GE Healthcare Australia Pty Ltd, Rydalmere, NSW, Australia).

96-well ELISA plates were coated with 10¹⁰ vp/100 µl/well of Wt-AdVs at 4°C overnight. The viruses were inactivated at 56°C for 30 min before the coating. After blocking with 200 μL of 10% skim milk at 37°C for 2 h, serially diluted serum samples were added and incubated at 37°C for 2 h. 1:5000 dilutions of horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (Sigma–Aldrich) was added and incubated at 37°C for 1 h. Subsequently, 50 μL of TMB substrate (New Cell & Molecular Biotech) was added to each well. Thereafter, 50 μL of 2 M H₂SO₄ solution was added to halt the reaction. Absorbance at 450 nm was measured using a Varioskan Flash multimode ELISA plate reader (Thermo Scientific). The antibody endpoint titre was determined as the highest serum dilution whose absorbance was 0.1 optical density (OD) unit above the control of PBS-treated samples.