American Society of Hematology

Figure 7

$Figure 7. Affinity-purified human anti-Neu5Gc antibodies bind specifically to Neu5Gc-loaded endothelial membrane glycoproteins and induce complement deposition. Affinity-purified human anti-Neu5Gc antibodies17 were used as a primary. (A) Biotinylated, affinity-purified human anti-Neu5Gc antibodies were studied by Western blotting against surface membrane glycoproteins of Neu5Ac (Ac)– or Neu5Gc (Gc)–loaded endothelial cells, and the banding pattern was compared with that generated with the cGcAb antibody. HUVECs loaded with either Neu5Ac or Neu5Gc were lifted with 100mM EDTA and sonicated. Membrane fractions were prepared, 3 μg denatured by boiling in SDS separated on 12.5% polyacrylamide mini gels (Bio-Rad), and electrotransferred to nitrocellulose membranes. Membranes were blocked overnight at 4°C with 0.5% Neu5Gc-free cold water fish gelatin (Sigma-Aldrich) in TBST. Primary antibodies were incubated for 5 hours at 4°C with either biotinylated anti-Neu5Gc antibodies purified from individual human sera, diluted 1:100 in TBST, or chicken anti-Neu5Gc antibody (cGcAb), diluted 1:50 000 in TBST. Purification of the cGcAb is described elsewhere.4 Membranes were washed 4 times for 5 minutes in TBST then incubated with Streptavidin-HRP (Bio-Rad), diluted 1:50 000, or HRP-anti-chicken-IgY (Jackson ImmunoResearch Laboratories), diluted 1:10 000, at room temperature for 45 minutes. Proteins were visualized by chemiluminescence detection (Pierce), followed by exposure to Kodak BioMax XAR film for 5 to 30 seconds. A representative example from 3 independently performed experiments is shown. (B) Anti-Neu5Gc IgG binding (x-axis) and complement deposition (y-axis) were simultaneously assessed on Neu5Ac- and Neu5Gc-loaded endothelium. Affinity-purified anti-Neu5Gc antibodies, low-titer human serum (S30), or both were incubated with Neu5Gc-loaded (top) or Neu5Ac-loaded (bottom) HUVECs. Although the low-titer human serum does not bind HUVECs in a Neu5Gc-dependent fashion (left), the affinity-purified anti-Neu5Gc antibodies are necessary and sufficient to bind Neu5Gc-loaded HUVECs in a Neu5Gc-dependent fashion (middle). Supplementing the low-titer serum with the purified anti-Neu5Gc antibodies allows complement deposition on HUVECs in a Neu5Gc-dependent manner (right). A representative result of 3 independent experiments is shown.$

Affinity-purified human anti-Neu5Gc antibodies bind specifically to Neu5Gc-loaded endothelial membrane glycoproteins and induce complement deposition. Affinity-purified human anti-Neu5Gc antibodies¹⁷ were used as a primary. (A) Biotinylated, affinity-purified human anti-Neu5Gc antibodies were studied by Western blotting against surface membrane glycoproteins of Neu5Ac (Ac)– or Neu5Gc (Gc)–loaded endothelial cells, and the banding pattern was compared with that generated with the cGcAb antibody. HUVECs loaded with either Neu5Ac or Neu5Gc were lifted with 100mM EDTA and sonicated. Membrane fractions were prepared, 3 μg denatured by boiling in SDS separated on 12.5% polyacrylamide mini gels (Bio-Rad), and electrotransferred to nitrocellulose membranes. Membranes were blocked overnight at 4°C with 0.5% Neu5Gc-free cold water fish gelatin (Sigma-Aldrich) in TBST. Primary antibodies were incubated for 5 hours at 4°C with either biotinylated anti-Neu5Gc antibodies purified from individual human sera, diluted 1:100 in TBST, or chicken anti-Neu5Gc antibody (cGcAb), diluted 1:50 000 in TBST. Purification of the cGcAb is described elsewhere.⁴ Membranes were washed 4 times for 5 minutes in TBST then incubated with Streptavidin-HRP (Bio-Rad), diluted 1:50 000, or HRP-anti-chicken-IgY (Jackson ImmunoResearch Laboratories), diluted 1:10 000, at room temperature for 45 minutes. Proteins were visualized by chemiluminescence detection (Pierce), followed by exposure to Kodak BioMax XAR film for 5 to 30 seconds. A representative example from 3 independently performed experiments is shown. (B) Anti-Neu5Gc IgG binding (x-axis) and complement deposition (y-axis) were simultaneously assessed on Neu5Ac- and Neu5Gc-loaded endothelium. Affinity-purified anti-Neu5Gc antibodies, low-titer human serum (S30), or both were incubated with Neu5Gc-loaded (top) or Neu5Ac-loaded (bottom) HUVECs. Although the low-titer human serum does not bind HUVECs in a Neu5Gc-dependent fashion (left), the affinity-purified anti-Neu5Gc antibodies are necessary and sufficient to bind Neu5Gc-loaded HUVECs in a Neu5Gc-dependent fashion (middle). Supplementing the low-titer serum with the purified anti-Neu5Gc antibodies allows complement deposition on HUVECs in a Neu5Gc-dependent manner (right). A representative result of 3 independent experiments is shown.

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