American Society of Hematology

Figure 3

$Figure 3. Binding of mAb H17 to PNH erythrocytes incubated in acidified NHS. Erythrocytes from patient 1 were incubated in aNHS chelated with EDTA (to prevent complement activation; NHS/EDTA, left panel), with aNHS (middle panel), or with aNHS containing 5 μg/mL fluorescently labeled mAb H17 (Al647 mAb H17, right panel). Unlysed erythrocytes and ghosts were separated by differential centrifugation. In the case of the cells initially incubated with NHS-EDTA or with aNHS, the unlysed and lysed populations were incubated with a combination of fluorescently labeled mAb 1H8 (Al488 1H8), mAb H17 (Al647 H17), and anti-CD59 (PE CD59). In the case of the cells initially incubated with aNHS containing 5 μg/mL Al647 mAb H17, the unlysed and lysed populations were incubated with a combination of fluorescently labeled mAb 1H8 (Al488 1H8) and phycoerythrin-labeled anti-CD59. Erythrocytes are denoted in gray histograms; ghosts, in solid histograms. (A) Erythrocytes and ghosts analyzed for bound C3 fragments based on probing with Al488 mAb 1H8. (B) Analysis for CD59 expression. (C) Analysis for binding of Al647 mAb H17. The percentage of erythrocytes (E) or ghosts (G) contained in the area indicated by the mark is shown in the upper right corner of each panel. (D) Ghosts analyzed for binding of both mAbs 1H8 and H17. Neither mAb 1H8 nor mAb H17 binds to intact erythrocytes after incubation in aNHS, indicating that the APC was not activated on these cells. The gate used to analyze antibody binding to ghosts includes some debris, likely accounting for the fraction of ghosts that are not opsonized with C3 fragments after reaction in aNHS, as this pattern is also present in the controls (NHS/EDTA). All samples were analyzed in duplicate, and 1 representative flow cytometry experiment is illustrated.$

Binding of mAb H17 to PNH erythrocytes incubated in acidified NHS. Erythrocytes from patient 1 were incubated in aNHS chelated with EDTA (to prevent complement activation; NHS/EDTA, left panel), with aNHS (middle panel), or with aNHS containing 5 μg/mL fluorescently labeled mAb H17 (Al647 mAb H17, right panel). Unlysed erythrocytes and ghosts were separated by differential centrifugation. In the case of the cells initially incubated with NHS-EDTA or with aNHS, the unlysed and lysed populations were incubated with a combination of fluorescently labeled mAb 1H8 (Al488 1H8), mAb H17 (Al647 H17), and anti-CD59 (PE CD59). In the case of the cells initially incubated with aNHS containing 5 μg/mL Al647 mAb H17, the unlysed and lysed populations were incubated with a combination of fluorescently labeled mAb 1H8 (Al488 1H8) and phycoerythrin-labeled anti-CD59. Erythrocytes are denoted in gray histograms; ghosts, in solid histograms. (A) Erythrocytes and ghosts analyzed for bound C3 fragments based on probing with Al488 mAb 1H8. (B) Analysis for CD59 expression. (C) Analysis for binding of Al647 mAb H17. The percentage of erythrocytes (E) or ghosts (G) contained in the area indicated by the mark is shown in the upper right corner of each panel. (D) Ghosts analyzed for binding of both mAbs 1H8 and H17. Neither mAb 1H8 nor mAb H17 binds to intact erythrocytes after incubation in aNHS, indicating that the APC was not activated on these cells. The gate used to analyze antibody binding to ghosts includes some debris, likely accounting for the fraction of ghosts that are not opsonized with C3 fragments after reaction in aNHS, as this pattern is also present in the controls (NHS/EDTA). All samples were analyzed in duplicate, and 1 representative flow cytometry experiment is illustrated.

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