FIG 2.

Determination of GP1/PS ratios in rLCMV-LASVGP and authentic LASV. (A) Schematic of the capture ELISA. Virus is captured by immobilized MAb 86.3, which recognizes a conserved nonneutralizing epitope in GP2. After removal of unbound material, bound virus is probed with ANX-V to detect PS, displayed in the lipid bilayer of the viral envelope, and DGEKFc4, which binds to GP1. For details, please see the text. (B) Detection of GP1 and PS. Purified rLCMV-LASVGP and authentic inactivated LASV were incubated with immobilized MAb 83.6, with the nonenveloped AdV5-GFP used as a negative control. After the plates were washed, bound virus was incubated with biotin-conjugated ANX-V in the presence and absence of EGTA, as well as DGEKFc4. Bound biotinylated ANX-V and DGEKFc4 were detected by streptavidin-HRP- and HRP-conjugated anti-human Fc antibody in a color reaction. Please note the reduced ANX-V binding in the presence of the Ca²⁺ chelator EGTA. Data are means ± SD (n = 3). (C) Ratios of GP1 to PS calculated for the virus preparations tested in panel B. OD (450), optical density at 450 nm; LASVi, inactivated LASV.