Human serum antibodies react against Neu5Gc-loaded endothelium. Human sera (S#), diluted 1:5, were assayed for their ability to bind HUVECs in a Neu5Gc-dependent manner. (A) Binding of human serum IgG (top) or IgM (bottom) to loaded HUVECs was assayed by flow cytometry. Neu5Gc-loaded cells (solid line), Neu5Ac-loaded cells (dotted line), and Neu5Gc-loaded cells stained with secondary antibody only (shaded curve). Representative individual human sera are shown. (B) IgG and IgM binding were normalized as a percentage value of the signal obtained with S34. The data are presented as mean ± SEM (n = 3). (C) Binding of anti-Neu5Gc IgG antibodies from S37 to Neu5Gc-loaded HUVEC was inhibited with the alpha-methyl glycoside, Neu5Gc2Me, in a dose-dependent manner and not by Neu5Ac2Me. (D) Classical complement, C3 convertase (C3b), was deposited onto Neu5Gc-loaded HUVEC (solid lines) only when incubated with high-titer anti-Neu5Gc serum (S34, right) and not low-titer anti-Neu5Gc serum (S30, left). Nonspecific complement was deposited onto Neu5Ac-loaded HUVEC (dotted lines) in a variable manner dependent on which sera was used, presumably attributable to unrelated serum factors. As a negative control, heat-inactivated serum was incubated with Neu5Gc-loaded HUVECs as a negative control (shaded curve). (E) Heat-inactivated S34 alone did not deposit complement in Neu5Gc-loaded HUVECs (shaded curve). Supplementation of S30 with heat-inactivated S34 shows Neu5Gc-dependent complement deposition in Neu5Gc-loaded (solid line) and not Neu5Ac-loaded HUVECs (dashed line). This finding indicates that anti-Neu5Gc antibodies are necessary and sufficient to mediate Neu5Gc-dependent complement deposition. All results presented in this figure were performed at least 3 times.