Figure 3

Neutralizing activity of natural (■) and recombinant (○) purified 26A1 IgG. The 26A1 mAb was purified from serum-free supernatants by protein A chromatography. The neutralizing activity of HCMV infectivity was then tested either with the laboratory strain AD169 in HELF (panel A) or with the clinical isolate VR1814 in HUVEC (panel B). The effect of 26A1 IgG on HCMV infectivity was measured by staining the HCMV IEA by indirect immunoperoxidase. Five fields (HPF 20×) were counted per well from triplicate wells. Percentage of inhibition of viral infectivity was calculated using the following formula: [(means ± s.d. of IEA⁺ nuclei of HCMV infected cells – means ± s.d. of IEA⁺ nuclei of 26A1-treated HCMV infected cells/means ± s.d. of IEA⁺ nuclei of HCMV infected cells) ×100]. Results represent the means ± s.d. of 3 independent experiments. Plaque reduction assay (panel C) shows the neutralizing activity of natural and recombinant 26A1 mAb against HCMV AD169 (1,000 pfu/rxn) in HELF. The values were plotted as a function of IgG concentration, and the concentrations of IgG required to inhibit viral infection by 50% (IC₅₀) were calculated by linear regression using GraphPad Prism (v. 4.0) software.