Effects of mAbs on hemolysis of PNH erythrocytes. Concentration-response effect of monoclonal antibodies (mAbs) H17 and 3E7. Erythrocytes from paroxysmal nocturnal hemoglobinuria (PNH) patients 1 and 2 were incubated in aNHS in the presence of incremental concentrations of the mAbs, and hemolysis was subsequently quantified. Experiments for separate blood samples obtained from these patients (all in 2009) in March (A,F), April (B-C, patient 2), and June (D, patient 1; E, patient 2) are shown. Sample standard deviations for duplicate or triplicate determinations are provided. In certain experiments only single points were evaluated, and in these instances, error bars are absent. (A) A concentration-dependent effect was observed for mAb H17 with near complete inhibition observed at a concentration of 170 μg/mL mAb H17. (B-E) Comparison of the effects of H17, 3E7, and Al488 H17 or Al488 3E7 on lysis in acidified serum. Similar dose responses for inhibition of lysis are evident. Each experiment illustrated in panels A through F is representative of at least 1 other comparable experiment. (F) Specificity of 3E7/H17. Erythrocytes from PNH patients 1 and 2 were incubated in aNHS in the presence of a fixed concentration of mAb, and hemolysis was subsequently quantified. The murine IgG₁ mAb 3E7, which is specific for C3b/iC3b and inhibits APC C3 convertase formation, and H17 (chimeric-deimmunized 3E7) block acidified serum lysis, but another murine IgG₁ mAb, 7C12, which recognizes an epitope expressed on C3b/iC3b distinct from that of H17/3E7, and a mAb of irrelevant specificity, 8B3 (mouse IgG₁), have no effect. (G) Normal human erythrocytes reacted with AET and additionally blocked with anti-CD55 mAb HD1A are lysed in aNHS, and mAb H17 blocks lysis. Representative of 5 similar experiments. (H) Effects of mAbs on CPC-mediated lysis of PNH erythrocytes. Erythrocytes from patient 2 were incubated with EDTA-chelated serum from a patient with CCAD to allow binding of the IgM antibody. After washing, the IgM-bearing cells were incubated with NHS and incremental concentrations of mAb H17 or 300 μg/mL mAb 5G9 (specific for the CPC). The results of 1 experiment in duplicate, representative of 3 experiments, are illustrated. The dashed line is set at the percentage lysis observed in the absence of mAb H17 (0 μg/mL). mAb H17 has no effect on hemolysis of PNH erythrocytes mediated by the CPC.