Activation of endothelial cells by affinity-purified human anti-β₂GPI IgG. Endothelial cells were cultured in 96-well plates and prepared as described in “Materials and methods.” Human anti-β₂GPI IgG was affinity purified from patients with antiphospholipid antibodies using Affigel HZ conjugated to β₂GPI. Endothelial cells were incubated for 4 hours with medium, β₂GPI (100 nM), affinity-purified (a.p.) human anti-β₂GPI IgG (600 nM), β₂GPI (100 nM) and affinity-purified human anti-β₂GPI IgG (600 nM), β₂GPI (100 nM) and nonimmune human IgG (NHIgG) (600 nM), or LPS (1 μg/mL). Endothelial cell surface E-selectin expression was then measured by ELISA. Error bars represent the mean plus or minus SEM of quadruplicate points. ***P < .0001 versus medium alone. This experiment is representative of 3 so performed using affinity-purified anti-β₂GPI from a single patient. Anti-β₂GPI IgG from 2 additional patients yielded similar results.