Figure 1.

Application of the FFA to detect HTNV titers and its performance compared with that of conventional methods. (A) HTNV CCID₅₀ titering by an ELISA-based method. Vero E6 cells in 96-well plates were inoculated with HTNV, maintained for 10 dpi, and subjected to three freeze/thaw cycles. Then, the supernatant was collected for titering. (B) Detection of the HTNV titer with the FFA method. Vero E6 cells in 12-well plates were inoculated with 10-fold-diluted HTNV and maintained with a CMC overlay. At 7 dpi, the FFA was performed with HRP-1A8 and AEC solution staining to assess HTNV NP expression. (C) Detection of the HTNV titer with the FFA method for a known CCID₅₀ titer at different timepoints. Vero E6 cells in 12-well plates were inoculated with HTNV and maintained with a CMC overlay. At 3 dpi, 5 dpi, and 7 dpi, the FFA was performed. (D) Western blot measurement of NP expression with 10-fold-diluted HTNV at 3 dpi. Vero E6 cells in six-well plates were inoculated with 10-fold-diluted HTNV and maintained for 3 dpi, and 1A8 was used as the detection antibody. (E) IFA of NP expression with 10-fold-diluted HTNV at 3 dpi. Vero E6 cells on coverslips in 24-well plates were inoculated with 10-fold-diluted HTNV and maintained for 3 dpi, and 1A8 was used as the detection antibody. (F) FFA detection of the HTNV titer with 10-fold-diluted HTNV at 5 dpi. These experiments were performed independently at least three times with similar results.