Figure 3

Verification of the binding activity of scFvs expressed by DF-1 to FJ13 virus. (A) Transient expression of scFvs in serum-free cultured DF-1 cells indirectly detected by EGFP. (B) Western blot assay used to directly detect the transient expression of scFvs (~30 KDa) in serum-free cultured DF-1 cells. (C) Indirect-ELISA assay used to assess the titer of scFvs binding to HA protein. (D) IFA used to detect the virus binding activity of scFvs. After infection by FJ13 virus (yellow) for 36 h, MDCK cells (red) were subjected to IFA using scFv1, scFv2, scFv3 and monoclonal antibody against AIV H5N1 subtype HA protein as primary antibodies. The positive control used the 1:300 anti-AIV H5N1 HA mAb and the negative used antibody diluent as the primary antibodies. (E) Virus neutralization of ScFvs, detected by IFA. The scFvs and anti-AIV H5N1 HA mAb (1:30, as the positive control) were incubated with FJ13 virus (200CCID₅₀, yellow ) respectively for 1h before infection of MDCK cells (red). The negative control used antibody diluent instead. Anti-AIV H5N1 HA mAb was used as the primary antibody. In (D) and (E), the cells were counterstained with PI (red). Data are from 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; *P <0.05; ** P <0.01; ns, not significant.