Antibodies cloned by single-cell sorting using cell surface–expressed gp160Δc^BaL. (A) Cell sorting strategy. Dot plot (left) shows percentage of doublets composed of GFP-293T^BaL cells and CD20⁺ B cells from one HIV-1–infected donor (patient 3B). IgG-expressing B cell/GFP-293T^BaL doublets (middle) were sorted into single wells and subjected to antibody cloning procedure. (right) ImageStream^X visualization of a PE-stained B cell (orange) attached or in close proximity to a larger GFP-positive gp160Δc^BaL-transfected 293T cell. (B) Pie charts depicting expansion of clonally related antibodies (colored) cloned from four HIV-1–infected individuals (3B, 7A, 8A, and C69). The numbers within the inner circles (top row) indicate the total number of IgH sequences analyzed. Percentages of clonally related sequences are shown in the middle row. Total numbers of antibody clones are displayed within the inner circle (bottom row) and expansion of clones are proportionally displayed in pie charts (bottom row). (C) mAbs were tested by FACS (y axis shows MFI) for binding to 293T cells transfected with GFP-harboring plasmids encoding gp160Δc^BaL (black bar), gp160Δc^YU2 (gray bar), or no-insert (white bar). All 15 antibodies that bound to gp160Δc^BaL are shown. PG16, b12 (positive controls), and mGO53 (negative control) were included. (D) The same antibody panel as in C was tested for binding to soluble HIV-1 proteins (gp140^BaL and gp140^YU2) by ELISA. Graphs show OD405nm (y axis) and antibody concentration in µg/ml (x axis). Binding analysis of generated antibodies to soluble protein (D) and cell surface–expressed gp160Δc^BaL/gp160Δc^YU2 (C) was at least performed in duplicates.