Lack of CD20 modulation on malignant B cells following rituximab treatment in vitro in the presence or absence of plasma. (A) Specificity of the mouse anti-rituximab idiotype (anti-Id) mAb, MB2A4. Following binding of various mouse/human chimeric mAbs (10 μg/mL) to EHRB or Raji cells for 15 minutes at room temperature (mAb [target]; 2B8 [mouse Ab against CD20]; rituximab [CD20]; chAT80 [CD20]; chWR17 [CD37]; chAT13/5 [CD38]; and chLOB7-4/7-6 [CD40]), cells were washed twice in phosphate-buffered saline/bovine serum albumin (BSA)/Azide before being stained with FITC-labeled mAb MB2A4 (10 μg/mL) for 15 minutes at room temperature and being analyzed by flow cytometry. As can be seen, binding of 2A4 was observed only on cells coated with rituximab or its parent mAb 2B8, demonstrating both the ability of the mAb to detect rituximab when it was bound to cells and its specificity for the rituximab V regions. (B) Typical dot-plots generated by staining B-CLL cells incubated with either rituximab or 2B8 in the presence or absence of 33% plasma for 2 hours at 37°C, followed by washing, as indicated in panel A, and staining with either FITC-labeled an ti-Id (2A4) or FITC-labeled anti-human Fcγ (Hu Fcγ, SB2H2) for 30 minutes at room temperature and analysis by flow cytometry. The anti-Id mAb was able to detect both mAbs equally well in the presence or absence of plasma, whereas the Hu Fcγ mAb could not detect rituximab after incubation in the presence of plasma. (C) The importance of wash steps in the detection of rituximab. After binding and incubation as in panel B, B-CLL cells were washed 1, 2, or 3 times by standard centrifugation prior to staining with either FITC-labeled anti-Id (MB2A4) or FITC-labeled Hu Fcγ as described in panel B. Flow cytometry data are expressed as the percent of FITC staining measured in the presence, compared with the absence, of 33% plasma. These data show that the detection of rituximab with anti-Hu Fcγ mAb was extremely sensitive to the number of wash steps used and that no apparent modulation of rituximab was observed when sufficiently washed. Conversely, the anti-Id mAb was unaffected by the number of wash steps. This effect of wash steps was replicated in 7 different experiments, using either Raji or B-CLL cells. (D) IgG in plasma blocks the detection of mouse/human chimeric mAbs. Raji cells were incubated with either rituximab or other chimeric human Fc-containing mAbs directed to CD20 (chAT80) or CD37 (chWR17), or with the parent mouse mAbs (2B8, AT80, or WR17). Cells were incubated for 2 hours at 37°C in the presence of media alone (NT), plasma, or plasma depleted of IgG (using protein A column chromatography), before was hing twice and staining with FITC-labeled anti-human or anti-mouse Fcγ. These data show that all mAbs with a human Fcγ domain showed apparent down-modulation in the presence of plasma, but that this “modulation” is not apparent when plasma was depleted of IgG or when the murine counterparts were used in the presence of human plasma.