FIG. 4.

ELISA binding curves of select MAbs against native and deglycosylated gp120_JR-FL. In the top panels, gp120_JR-FL was either treated with endoglycosidase H (EndoH, solid squares) or jack bean mannosidase (JBM, solid triangles) or left untreated (open diamonds) overnight at 37°C, then captured via the C-terminal region of gp120 by using the sheep Ab D7324, and probed with 4KG5, 2G12, and Fab b12. In the bottom panels, gp120_JR-FL was immobilized in a microplate well and then treated with sialidase for 2 h at 37°C followed by β-galactosidase for 28 h at 37°C (closed triangles) or with sialidase alone (closed squares). Untreated samples (open diamonds) were included as controls. These samples were probed with 4KG5, hNM01, and 2G12. The detection reagent for 4KG5 was ABTS (top panel) or TMB (bottom panel), and AP staining solution was used for the other panels. The OD was read at 450 nm for 4KG5 (bottom panel only) and 405 nm (or all other panels).