Comparison between antibody binding to yeast surface displayed gp140 and gp140 purified from mammalian cells.

Unmodified YU2 gp140 and YU2 gp140dsm, containing stabilizing mutations (see text), were expressed as a fusions at their C-termini to Aga2p. Alternatively, purified, biotinylated YU2 gp140 (17 nM) was bound (at a saturating concentration) to yeast cells displaying a streptavidin-Aga2p fusion protein. Binding was detected by flow cytometry following incubation with fluorescently tagged secondary antibodies (Antibody concentrations were: 70 nM (anti-Env antibodies); 20 nM (anti-V5 tag antibody). The indicated values are the ratios of the mean fluorescence intensities for the relevant anti-HIV antibodies to the mean fluorescence intensity for binding of anti-V5 epitope antibody to each yeast strain (measured in the absence of purified biotinylated gp140). Fluorescence intensity for binding of anti-V5 epitope antibodies to the streptavidin-Aga2p fusion (in the absence of purified biotinylated gp140) was 7.5 (± 0.4, n = 6)-fold higher level than the level for binding of anti-V5 to yeast-displayed YU2 gp140 fused to Aga2p and 2.5 (± 0.1, n = 6)-fold higher level for anti-V5 binding to YU2 gp140dsm fused to Aga2p (data not shown). Secondary antibody incubation was performed at 4°C and flow cytometry was performed with cells kept on ice.