Figure 5

Comparison of plaque neutralization and VRP based NT assays. (A) CHIKV infected and non-infected (mock) BHK cells were used for indirect immunofluorescence analyses using the indicated monoclonal antibody or human sera. Nuclei were stained with DAPI. The bar represents 25 μm. (B) For the plaque neutralization assay, serial dilutions of monoclonal antibody D3.62 or three patient sera were preincubated with infectious CHIKV. NT assays were performed in 6-well format with readout at 48 h p.i. using crystal violet staining. The bar labeled with Virus represents a non-neutralized infectivity control, which was set 100%. The % infectivity was normalized to the non-neutralized virus infection. The gray dotted line indicates the 50% infectivity threshold. Data represent the means and ranges of duplicate infection experiments. (C) For the VRP based NT assay, serial dilutions of monoclonal antibody D3.62 or three patient sera were preincubated with VRPs. NT assays were performed in either 24-well plates (top panel) or 96-well plates (bottom panel) using VRPs at an MOI of 5. Readout was performed at 6 h p.i. via measurement of Gluc secreted into the supernatant. The bar labeled with VRP represents infection with the appropriate amount of VRPs not preincubated with patient sera/antibody. Negative (Neg.) serum from a person not infected with CHIKV was used as control. The % infectivity was normalized to VRP infection without serum/antibody incubation. The gray dotted line indicates the 50% infectivity threshold. Data represent average and standard deviation of experiments performed in triplicate.