Fitc conjugated rabbit anti pig igg

Nanobodies were conjugated with fluorescein isothiocyanate (FITC) using a FITC Conjugation Kit (Bioss, China) according to the manufacturer’s instructions. Primary PAMs were seeded in glass bottom cell culture dishes (NEST, China) at a density of 4.0 × 10⁶ cells and infected with ASFV at an MOI of 1.0. At 48 h post-infection, ASFV-infected or negative control cells were fixed with 4% paraformaldehyde for 10 min, permeabilized in 0.1% Triton-X 100 for 10 min, and blocked with 5% BSA for 1 h at RT. The fixed ASFV-free and -infected PAMs were washed three times with PBS and incubated with FITC-conjugated nanobodies for 1 h at 37°C. Moreover, ASFV-infected PAMs were also incubated with ASFV-positive and -negative serum for 1 h at 37°C, respectively. After washing three times with PBS, the FITC-conjugated rabbit anti-pig IgG (Sigma, United States) was added and incubated for 1 h at 37°C. Then, cells were incubated with DAPI (Invitrogen, United States) for 2 min at RT and rinsed with PBS. The cells were visualized for immunofluorescence with a fluorescence microscope (Leica, Germany).

The highly virulent CSFV Shimen strain was obtained from the Institute of Veterinary Drug Control, China. Positive anti-CSFV serum and negative control serum were prepared as described previously [30 (link)]. Freshly trypsinized fibroblasts of the Mx1 transgenic pigs and age-matched controls were added to 24-well plates and incubated at 39°C in 5% CO₂. When 80-90% confluent, cells were infected with the CSFV Shimen strain (400 TCID₅₀ per well). At 60 hours post infection, the culture media were aspirated and the cell monolayers in wells were fixed with 80% cold acetone for 30 minutes, washed 3 times with PBS, and incubated for 1 h with pig anti-CSFV serum (1:100 dilution) at 37°C in a humidified box. Cells were then washed 3 times with PBS and incubated with FITC-conjugated rabbit anti-pig IgG (1:60 dilution, F4762; Sigma) for 1 hour. After thoroughly washing, positive cells were photographed and counted under an inverted fluorescence microscope. The FITC fluorescence of the secondary antibody stain could be observed clearly using a 4 × objective lens and there was no interference of the EGFP signal in the transgenic cells at this amplification level.

These assays were performed according to Xu et al. [18 (link)]. CSFV (SM) from the Institute of Veterinary Drug Control, China, was used in this study. Positive anti-CSFV serum was kindly provided by Dr. CC Tu. To determine the viral titer, cells were infected with CSFV for 60 h, and samples of the medium were collected, serially diluted 10-fold from 10^-1 to 10^-6 and inoculated onto PK-15 cells. Each dilution was examined for viral proliferation using IFA. The TCID₅₀ values were calculated using the Kärber method. For IFA, cells were infected with CSFV for 72 h, fixed in precooled 80 % acetone, and incubated with the positive anti-CSFV serum. A FITC-conjugated rabbit anti-pig IgG (Sigma) was used as the secondary antibody, and the cells were examined using an ECLIPSE TE2000-V (Nikon). Real-time PCR was performed with an iQ^tm5 Multicolor Real-time PCR Detection System (Bio-Rad) using a QuantiTect™ Probe PCR Kit (QIAGEN). The special primers (rFPprimer, rRPprimer and Prob5B) for the CSFV NS5B gene have been previously described [14 (link)].