FIG. 4.

Dose-dependent inhibition of SVP binding to DF-1 cells by MAbSVP-4, a neutralizing MAb. (A) MAbSVP-4 protects CEF cells from IBDV infection. The IBDV neutralization capabilities of MAbSVP-4, MAbSVP-1, and anti-VP2 were measured by virus neutralization assays. Serum samples were diluted twofold, starting at a 1:16 dilution, in Medium 199 and mixed with 200 TCID₅₀ of IBDV P3009 per well (final serum dilution, 1:10,240) at 37°C for 1 h. The antibody-virus mixture was then added to 2 × 10⁴ CEF cells in Medium 199 containing 10% fetal calf serum per well in a 96-well culture plate. After 4 days at 37°C, cell monolayers were observed under a microscope for CPE and were then washed with PBS and stained for 20 min with 1.5% crystal violet in 50% ethanol. The titer of neutralizing activity was evaluated by visual screening of the infected monolayers, and the end point was calculated as the reciprocal value of the highest serum dilution that causes a 50% reduction of the cell monolayer. Results for MAbSVP-4 with a dilution factor of 1,024 and for MAbSVP-1 with a dilution factor of 16 (same as anti-VP2) are shown. CPE of cells incubated with a mixture of MAbSVP-1 (or anti-VP2) and virus could be observed as clearly as that in the negative control, where CEF cells were infected with only 200 TCID₅₀ of the virus. A similar result was not shown for the cells treated with a mixture of anti-VP2 and virus. In contrast, with MAbSVP-4 neutralizing IBDV and protecting the cells from the infection, cells incubated with MAbSVP-4 and virus complex were as healthy as cells of the control group (top). (B) Flow cytometry histograms. Shown are staining DF-1 cells with FITC-conjugated SVP preincubated with either different concentrations of MAbSVP-4 or anti-VP2. Violet, control cells; blue, cells incubated with FITC-labeled SVP; red, cells incubated with FITC-labeled SVP and MAbSVP-4; green, cells incubated with FITC-labeled SVP and anti-VP2 polyclonal antibody.