IVIg contained antibodies against human Siglec-9 and bound to neutrophils, as assessed by surface plasma resonance (SPR) and flow cytometry. (A) Sensorgrams of the binding activity of different fractions of IVIg (all at 1 μM) and a control anti–Siglec-9 mAb (1 nM) to immobilized recombinant Siglec-9/Fc fusion protein are shown. Binding of total IVIg to Siglec-9 was detected, confirming the existence of autoantibodies against Siglec-9 in IVIg (anti–Siglec-9 IVIg). In contrast, anti–Siglec-9–depleted IVIg did not show any binding to Siglec-9, suggesting that autoantibodies against Siglec-9 were successfully depleted from this fraction. In comparison, affinity-purified anti–Siglec-9 IVIg showed a marked increase in the association curve. (B) Freshly isolated mature blood neutrophils were analyzed by flow cytometry. Purified human anti–Siglec-9 Abs and mouse anti–Siglec-9 mAb (clone E10-286) similarly bound to the cell surfaces of neutrophils (black peak: control staining; white peak: anti–Siglec-9 staining). Representative examples of 4 independent experiments are shown.