aRh17 and aWr^b antibodies demonstrate differential effects on RBC resistance to osmotic and mechanical stress. Osmotic stress was induced by incubation in buffered (10 mM sodium phosphate) saline at a range of osmolalities (0-308 mOsm). Mechanical stress was induced by rotation in the presence of glass beads at 1% hematocrit (Hct). Antibodies were added at 10 nM and 100 nM to 5% Hct RBC suspension, which produces a ratio of ∼10⁴ and 10⁵ ligands per RBC and is below saturation for both target antigens. (A) RBCs treated with 500 nM aWr^b scFv (blue) showed a left shift in the osmotic lysis curve compared with naive (green) or aRh17 scFv-treated RBCs (red). (B) RBCs treated with aWr^b but not aRh17 showed a significant change in the concentration required for 50% hemolysis (128 vs 120 mOsm, n = 3, *P < .05, 1-way analysis of variance [ANOVA] with Holm-Sidak correction for multiple comparisons). (C) aWr^b scFv-treated RBCs (blue) show a dose-dependent decrease in hemolysis in response to osmotic stress at 128 mOsm (EC50 for naive RBCs) and (D) a dose-dependent increase in hemolysis in response to mechanical stress. aRh17 scFv-treated RBCs (red) do not demonstrate any significant change in response to (E) osmotic stress or (F) mechanical stress. In all experiments, means ± standard deviation (SD) are shown; n = 3 for each condition (*P < .05 compared with naive, 1-way ANOVA with Holm-Sidak correction for multiple comparisons). ns, not significant.