1072 Appl Microbiol Biotechnol (2010) 86:1067–1075 Fig. 3 HTNV GP antigen-specific binding characteristics of scFv3G1. a Solid-phase binding of purified scFv3G1 to HTNV GP antigen as measured by ELISA. The wells were coated with HTNV GP or HTNV NP and then incubated with purified twofold serially diluted scFv3G1 from 1,000 to 0.1 nM. b To compare the HTNV GP- specific antigen-binding activity of scFv3G1 to that of MAb3G1, serially diluted scFv3G1 and MAb3G1 were assayed. Results are on the fact that MAb3G1 is an IgG possessing two identical antigen recognition surface, while scFv3G1 retains only one. We assayed the immunoreactivity of scFv3G1 by Western blot and found that scFv3G1 detected a polypeptide band of about 56 kDa corresponding to HTNV glycoprotein G2 molecular weight similar to that observed with MAb3G1, while no staining background was detected with unrelated HTNV NP antigen and when irrelevant control HBsAg- specific scFv15 was used instead of scFv3G1 (Fig. 2c). To evaluate whether the scFv3G1 was able to bind HTNV GP on the cell membrane, Vero E6 cells infected with HTNV for 10 days were incubated with the purified scFv3G1 and examined by flow cytometry. This scFv bound with HTNV GP expressed on the membrane of HTNV-infected cells similar to that observed with MAb3G1 (Fig. 3c), while scFv3G1 did not react with uninfected cells, and several other cell lines (data not shown) and control HBsAg-specific scFv15 did not react with HTNV-infected cells in parallel plotted as percentages of HTNV GP binding. c Flow cytometric analysis to evaluate whether scFv3G1 can bind HTNV GP antigen expressed on the cell membrane. Ten-day HTNV-infected Vero E6 cells or uninfected cells were incubated with 100 nM purified scFv3G1 and followed by detection with FITC-conjugated anti-His- Tag antibody experiments (Fig. 3c). These results indicate that this recombinant scFv3G1 possesses biological activity of HTNV GP-specific binding. scFv3G1 can be internalized into cytoplasm of HTNV-infected cells Since intracellular delivery requires a cell type-specific internalizing antibody, we analyzed whether this scFv can be internalized into infected cells. Vero E6 cells preinfected with HTNV were incubated with FITC-tagged scFv3G1 antibody or FITC-tagged MAb3G1 for 30 min. As shown in Fig. 4a, specific fluorescence was observed both on the membrane and in the cytoplasm of HTNV-infected cells. This fluores- cence diffusion pattern indicates that both the scFv3G1 and the parental MAb3G1 can be efficiently internalized into the cytoplasm following binding to its target antigen HTNV GP on the surface of HTNV-infected cells.