Horseradish peroxidase hrp conjugated anti mouse igg antibody

1x10¹⁰ viral particles of Ad5 were incubated with 500 μg m9C12-Fab fragments, or PBS control for 30 min at RT. Mice were infected with virus +/- Fab by lateral tail vein injection. Blood samples were collected via the tail vein at 7, 14 and 21 days post infection. Serum samples obtained via centrifugation of clotted blood samples were used to determine the presence of anti-adenovirus specific antibodies by Elisa. Briefly, 96-well polystyrene microtiter plates (Microlon, Greiner) were coated overnight at 4°C with Ad5 (5x10⁹ pts/mL) in PBS. Plates were washed three times with 0.05% Tween 20 in phosphate buffered saline (PBS-T) before blocking in 5% skimmed milk-PBS- T for 1 h at 37°C and then three PBS-T washes. Plates were then incubated for 1h at 37°C with 1:200 dilution of each serum sample in duplicate in 5% skimmed milk-PBS-T. After three washes with PBS-T, 50 μl of horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Sigma Aldrich) diluted 1:1000 in 5% milk PBS–T, was added to each well and incubated at 37°C for 1h. The plates were washed four times with PBS-T and bound antibody detected with 50μl tetramethylbenidine (TMB, Invitrogen) followed by incubation at room temperature for 10 min. The reaction was stopped with 1 M H₂SO₄ and the optical density (OD) was read at 450 nm using a BMG PHERAstar FS plate reader.

Cellular extracts were separated by electrophoresis on 10% polyacrylamide gels and transferred to Hybond-P polyvinylidene difluoride membranes (Sigma-Aldrich) that were blocked afterwards with 5% nonfat dried milk (OXOID, Hampshire, United Kingdom) in phosphate buffer saline (PBS) for 1 h. Monoclonal antibody V9, specific for human vimentin (Sigma-Aldrich), was used for identification at 6.8 µg/mL. Anti-β-actin (Sigma-Aldrich) (3.1 µg/mL) or anti-G3PDH (1 µg/mL) antibodies were used as loading controls. All antibodies were diluted in PBS containing 5% nonfat dried milk and incubated for 1.5 h. A horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Sigma-Aldrich) at 8.8 µg/mL was used as secondary antibody. The activity of the HRP enzyme was visualized after 1 min incubation with 0.5 mg/mL diaminobenzidine (Sigma-Aldrich) in PBS, in the presence of 0.015% hydrogen peroxide (Merck, Darmstadt, Germany). Quantification of bands from Western blots was done using Image J software.