FIG. 7.
Monoclonal antibodies against the ISP neutralize its IFN-inducing activity and recognize gp67. (A) Serial dilutions of hybridoma supernatants were incubated in 96-well V-bottom plates with 0.2 μg of ISP per ml for 1 h at 37°C in a total volume of 125 μl of D−10+NEAA. This mixture was then added to 1.5 × 105 RAW264.7 cells/well (75-μl volume) in 96-well flat-bottom plates. After 8 h at 37°C, 100 μl of the supernatant was removed for IFN-α/β ELISA. The control antibody (Ab) used is a hamster monoclonal antibody which reacts with bacterial glutathione S-transferase. (B) Cultured media from Sf9 cells infected with baculovirus (BV) or control media from uninfected cells (200 μl) or cell lysates from Sf9 cells transiently transfected with the AcNPV gp67 construct were immunoprecipitated with 10 μg of purified ISP-6E11. Immunoprecipitates were subjected to 10% SDS-PAGE in the presence of 2% 2-ME and transferred to nitrocellulose. Membranes were blotted with biotinylated ISP-6B12 (0.5 μg/ml).