GL-2045 sequesters C1q and prevents deposition of C1q, C4b, and MAC on the surface of Ab-opsonized cells. (A) The ability of testing compounds binding to plate-coated C1q was measured by ELISA. (B) GL-2045 blocks RTX-mediated C1q, C4b, and MAC deposition on Ramos cells in a CDC assay. GL-2045, HAGG, or IVIG was incubated in NHS and added to RTX-opsonized Ramos cells. Incubations were carried out for 15 minutes for C1q and C4b deposition or 30 minutes for MAC formation. The cells were then stained with FITC-conjugated anti-C1q, anti-C4b, or anti-C5b-9 mAbs to examine C1q, C4b, or MAC deposition. Data are shown as percent of C1q-, C4b-, or MAC-positive cells within the total cell population. (C) GL-2045 inhibits purified C1q deposition on RTX-opsonized cells. GL-2045, HAGG, or IVIG was preincubated with purified C1q (20 μg/mL) for 10 minutes and the resultant mixture was incubated with RTX-opsonized SUDHL4 cells at 37°C for 15 minutes. C1q deposition was detected with FITC anti-C1q Ab and evaluated by flow cytometry. Data are shown as percentage of C1q-positive cells within the total cell population. (D) GL-2045 inhibits the activation of the classical complement pathway in a plate-based assay. HAGG was coated on a plate to activate the classical pathway. Test compounds were added to the wells and incubated with 1.5% NHS at 37°C for 30 minutes for C1q deposition or 1 hour for MAC deposition. C1q and MAC depositions were determined by ELISA. The results are shown as the least squares mean ± SE estimated by ANCOVA. Natural log variance stabilizing transformation and Tukey procedure for multiple comparisons adjustment were used to test the differences. *P < .05, **P < .01, ***P < .001 compared with no-treatment control.