Figure 2

Biotinylation of secretory scFv proteins. Western blot gel retardation assay of supernatants from cells transiently transfected (A) or stably transfected (B) with the bigenic vector expressing the indicated scFv. (C) Supernatants from stably transfected cells taken at different culture times. Where indicated, cell cultures were supplemented with biotin for 24 h and dialyzed samples reacted with streptavidin. (D) ELISA of biotinylated or non-biotinylated scFv^P3 (coating chimeric mAb 1E10) and scFv^1E10 (coating chimeric mAb P3); binding was revealed with anti-SV5 tag mAb or HRP-conjugated-streptavidin. (E) Flow cytometry and (F) immunofluorescence microscopy of biotinylated scFvs on cells expressing specific cell-surface ligands. scFv^P3 (left panels) was reacted with cells displaying idiotype 1E10; scFv^9E1 (right panels) was reacted with RBL-SX38 expressing human FcεRI. Binding was performed with biotinylated (red) or non-biotinylated (violet) scFvs and with Quantum Dots-conjugated streptavidin. The green signal corresponds to FITC-conjugated anti-human IgE (ε-chain specific) antibody that recognizes the ε-membrane SIP displaying the idiotype.