Dissociation of FITC-conjugated F(ab′)2and Fab′ fragments of MoAb HB57 (A) or MoAb Mu53 (B) from Ramos cells in the presence or absence of excess unlabeled antibody. Cells were incubated with FITC-conjugated F(ab′)2 (○, •) or Fab′ (▵, ▴) fragments of high-affinity MoAb HB57 (A) or intermediate affinity Mu53 (B) (16.5 μg/mL; MoAb:cell ratio equal to that used for tyrosine phosphorylation experiment in Fig 5, ie, 5 μg/105 cells) for 5 minutes before addition of an excess of unlabeled MoAb HB57 diluted in sheath fluid or the addition of sheath fluid alone, as described in Materials and Methods. Flow cytometric analysis of the level of FITC-fluorescence was begun immediately after the addition of sheath fluid with or without unlabeled protein, using time as a parameter, for a total of 560 seconds. The amount of specific fluorescence at each 80-second time interval (t = x) was determined as described in Materials and Methods and is plotted above as a ratio of the specific fluorescence during each time interval relative to the specific fluorescence noted in the same tube at t = 0 to 20 seconds (ie, t = 0). At this latter time interval, there was no notable difference between the engagement of ligand to cells with or without unconjugated MoAb present (data not shown).
