Figure 8

Intracytoplasmic expression of IgG1 antibody delivered by adenovirus vector. Left panel shows dose-dependent intracytoplasmic expression of IgG1 antibody in Huh7.5 cells delivered by adenovirus vector. Huh-7.5 cells were infected with increasing concentrations of Ad-IgG. After 24 hours, intracellular antibody expression was examined by immunostaining with a biotin conjugated goat anti-human IgG1 antibody. Slides were then counterstained with hematoxylin. 1: Mock infected cells showing negative staining. 2-4: Dose dependent intracytoplasmic brown staining was seen in majority of Huh-7.5 cells infected with Ad-IgG virus (A). Western blot analysis showing the presence of light chain and heavy chain antibody in the lysates of Huh-7.5 cells infected with Ad-IgG (B). Huh-7.5 cells were infected with three different batches of recombinant Ad-IgG at a concentration of 4000 MOI. Cell lysates were made after 24 hours. 50 μg of protein lysates were separated on a 10% SDS PAGE and transferred to a nylon membrane. Immunoblotting was performed with peroxidase conjugated rabbit anti-human antibody. The membrane was developed using the ECL kit and was exposed to chemiluminescent-sensitive film. 1. Uninfected Huh-7.5 cells. 2. Huh-7.5 cells infected with an adenovirus luciferase (Ad-Luc). 3-5. Huh-7.5 cells infected with three different batches of plaque-purified virus (Ad-IgG). Two specific bands at molecular weight 27 kD and 52 kD represent the light chain and the heavy chain of the IgG1 antibody respectively.