Development of a sensitive flow cytometry assay to detect the binding of human antibodies to primary B-CLL cells. PBMCs (5 × 105, consisting of > 85% B-CLL cells) from a patient with untreated B-CLL were first incubated with normal goat serum to block Fcγ receptors (CD32B) followed by unconjugated goat Fab anti–human IgG pAbs to block cell-surface Ig (blocking). In the pilot experiment shown here, chimeric mouse/human anti–human CD20 mAb rituximab was the primary antibody that served as positive control for serum antibodies with human constant domains and cell-surface reactivity. Goat F(ab′)2 anti–human IgG pAbs conjugated to Qdot 655 nanocrystals (gαh-Qdot) were used as secondary antibodies. Substantial reactivity noted for the secondary antibody alone (A) was eliminated through cell-surface blocking (B), permitting detection of the primary antibody reactivity (C). The numbers inside each panel depict median fluorescence intensity (MFI).
