Journal: Virology Journal

Article Title: Attenuation of vaccinia virus by the expression of human Flt3 ligand

doi: 10.1186/1743-422X-7-109

Figure Lengend Snippet: Incorporation of sFL into viral particles . Aliquots of the indicated sucrose purified virions (A) or their solubilized and separated fractions (B) were analyzed by western blot using the FL specific BAF308 monoclonal antibody. The sample prepared from the culture medium of 293T cells transfected with pBSC-FL expression plasmid served as a positive control. To neutralize virus infectivity, the saccharose purified particles were incubated at 37°C for 1 hour with PBS, anti-FL antibody or anti-VACV serum. The first aliquot of samples was used for infection of cell cultures (C) . Cytosine arabinoside (40 μg/ml) was added for inhibition of viral replication as the positive control. The beta-galactosidase assay was performed after 24 hours of cultivation. The second aliquot of samples was applied to a formvar membrane coated copper grid (D) . Negatively stained particles were examined under an electron-microscope at a magnification of 50 000×.

Article Snippet: FL was quantified with an Flt3 ligand ELISA detection kit (R&D Systems GmBH, Wiesbaden-Nordenstadt, Germany) using the capture mouse monoclonal antibody MAB608 (100 ng/well), biotinylated detection goat polyclonal antibody BAF308 (7.5 ng/well), streptavidin-HPR (1:250) or avidin-HPR (1:1000) complex, both obtained from Pharmingen (BD Biosciences, Erembodegem, Belgium), and TMB substrate solution for visualization of the reaction.

Techniques: Purification, Western Blot, Transfection, Expressing, Plasmid Preparation, Positive Control, Infection, Incubation, Inhibition, β-Gal Assay, Staining, Microscopy