Inhibition of C3 deposition on PNH erythrocytes by mAb 3E7/H17. PNH erythrocytes from patient 2 were incubated in aNHS chelated with EDTA (to prevent complement activation; NHS/EDTA, left panel), with aNHS (45% lysis; middle panel), or with aNHS containing 150 μg/mL mAb H17 or 3E7 (4% and 5% lysis, respectively; right panels). Both lysed and unlysed cells were recovered by high-speed centrifugation, washed, and incubated with a combination of fluorescently labeled 1H8 (Al488 1H8; Table 1), a mAb that recognizes an epitope expressed on C3b, iC3b, and C3dg and phycoerythrin-labeled anti-CD59 (PE CD59). Gating based on forward scatter and side scatter characteristics was used to analyze the unlysed and lysed (ghost) cells separately. Erythrocytes are denoted in gray histograms; ghosts, in solid histograms. The percentage of ghosts in the samples was as follows: 5% in the sample incubated in NHS-EDTA; 31% in the sample incubated in aNHS; 9% in the sample incubated in aNHS containing 150 μg/mL mAb H17; and 17% in the 3E7 sample. (A) Binding of C3 activation and degradation products on the unlysed cells (erythrocytes) and on the ghosts. (B) Expression of CD59 on the unlysed cells and on the ghosts. The percentage of erythrocytes contained in the area indicated by the mark is shown in the upper right corner of each panel. The results of 1 experiment, representative of 5 experiments, are illustrated. After incubation in aNHS, CD59-deficient (low or absent) cells are hemolyzed and C3 fragments are deposited on the CD59-deficient ghosts (aNHS; A-B), and these processes are inhibited by both mAb H17 and 3E7.