1070 primary antibody solutions were scFv3G1, MAb3G1, or HBsAg-specific scFv15 twofold serially diluted in PBST from 1,000 to 0.1 nM. The plates were incubated with primary antibody solutions overnight at 4°C. The wells were washed again, and the bound antibodies were detected using either HRP-conjugated anti-His-Tag antibody or HRP-conjugated goat anti-mouse IgG in PBST at room temperature for 90 min. After final washing, 100 µL of substrate solution composed of 4 mg o-phenylenediamine (Sigma-Aldrich) in 10 mL of 0.1 M citrate–phosphate buffer (pH 5.0) containing 0.01% H2O2 was added to each well. The color reaction was developed in the dark for 15 min, and absorbance at 450 nm was determined using Tecan GENios microplate reader. Assay results are expressed as the means±SD of three independent experiments. Preparation of infected cells Cultures of Vero E6 cells were inoculated with HTNV strain 76–118 at a MOI of 0.5 in serum-free medium. After incubation for 1 h at 37°C, unbound viruses were removed by a triple washing. Maintenance medium (DMEM with 2% FBS) was added to the cells and changed every 5 days. The cells were ready for further use 10 days after inoculation. Biosafety level 3 facilities were used throughout these experiments for Hantavirus cultivation. Flow cytometry Flow cytometric analysis was used to evaluate whether scFv3G1 can bind HTNV GP expressed on the cell membrane. The 10-day infected Vero E6 cells or uninfected cells were washed, scraped, and incubated with the recombinant scFv3G1 at 100 nM in PBST at 4°C for 30 min. In parallel experiments, MAb3G1 was used as positive control, while HBsAg-specific scFv15 antibody acted as negative control. Cells were washed again and stained with fluorescein isothiocyanate (FITC)-conjugated anti-His-Tag antibody (Abcam) or FITC-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology) at 1:1,000 dilution in PBST at 4°C. After 30 min of staining, the cells were washed a third time and then analyzed by flow cytometry using a FACSCalibur (Becton Dickinson) instrument. Immunofluorescence staining The immunofluorescence staining was performed to deter- mine whether the scFv3G1 can be internalized into HTNV- infected cells. The Vero E6 cells infected with HTNV for 10 days were seeded on 24-well coverslips at 3× 104 cells per well and grown in DMEM (2% FBS) at 37°C in 5% CO2 incubator overnight. The cells were washed three Appl Microbiol Biotechnol (2010) 86:1067–1075 times in PBS and incubated with FITC-tagged scFv3G1, FITC-tagged MAb3G1, or together with Texas red-tagged transferrin (Molecular Probes) for 30 min at 37°C. Then, cells were washed and fixed in 3% paraformaldehyde for 5 min, followed by DAPI counterstaining for nuclei. After being washed with PBS, the coverslips were mounted on slides with ProLong Gold antifade reagent (Molecular Probes) and observed with a laser scanning confocal microscope (Olympus FV-1000). Accession numbers The nucleotide sequence for VH domains of the MAb3G1 is deposited at GenBank as FJ751231 and that for VL domains of the MAb3G1 as FJ751232. Results Cloning of the scFv3G1 coding sequence In order to investigate the cell type-specific binding and internalizing properties by HTNV-infected cells, coding sequences of VH and VL domains of the MAb3G1 were cloned initially from the parental hybridoma cell line and sequenced. The deduced amino acid sequences were compared with those of variable regions of immunoglobulins stored in the immunological Kabat database. The three loops corresponding to the CDRs of the VH and VL regions and the amino acid residues involved in the canonical structures were identified. The cDNA sequences of VH and VL are submitted to the GenBank database (accession number FJ751231 and FJ751232). The cDNAs encoding for MAb3G1 VH and VL were fused by splice-overlapped extension. PCR amplifica- tion using primers VHRevEcoRI and VLForSalI generated a 0.7-kb fragment (Fig. 1a). This PCR fragment was inserted between EcoRI/SalI cloning site in the pET32a vector and sequenced. Nucleotide and deduced amino acid sequences of scFv3G1 are shown in Fig. 1b. Production and purification of the scFv3G1 The recombinant protein was expressed by E. coli BL21 (DE3) strain transformed with pET32a-scFv3G1. Briefly, transformed BL21 cells were grown in LB medium at 37°C and incubated with 0.2 mM IPTG at 30°C for 3 h. Cytoplasmic recombinant protein, containing a thioredoxin (11 kDa) at the N terminus and two group of polyhistidines, was extracted after sonication. Immobilized metal affinity purification of scFv3G1 was carried out using Ni-NTA HisBind Resin that specifically binds the polyhistidine. Eluted fractions were collected and concentrated by ultrafiltration.