Functional analysis of CR1 derivatives in vitro. RBCs, sensitized with 6 different complement-fixing antibodies, were incubated with fresh sera as source of complement, which had been pretreated with mock (lane 1) or known complement inhibitors: either 10 mM EDTA (lane 2) to block all complement activation pathways or 10 mM EGTA in the presence of 2 mM MgCl2 (lane 3) to block the classical pathway; or CR1 derivatives all at a final concentration of 1 μM: LHR-A (lane 4), LHR-B (lane 5), LHR-C (lane 6), LHR-D (lane 7), LHR-D+ (lane 8), and sCR1 (lane 9). Hemolysis (hemoglobin release) in the supernatants was measured spectrophotometrically at 405 nm. The reactivity of remaining RBCs with anti-C3 using FITC-conjugated human anti-C3, or anti-C4 using FITC-conjugated human anti-C4, was measured by flow cytometry. Data are presented as percentage of control (Mock) hemolysis (▪), C4 (□), or C3 (▦) reactivities. Each series of experiments with a given antibody-sensitized antibody was repeated at least 2 times so that each bar represents the mean ± standard error of the mean (SEM) of least 12 experiments.
