CFHR1 but not CFH binds to C5. (A) CFHR1 was captured to the surface of a microtiter plate using the C-terminal (C18) or the N-terminal (JHD10) binding mAbs. C5 or C5b6 was applied with the fluid phase, and bound proteins were detected with mAb to C5. Attachment of CFHR1 to C18 or JHD10 was verified with polyclonal CFHR1 antiserum (columns 4 and 8). In addition, binding of C5b6 to the mAb JHD10 in the absence of CFHR1 was assayed and was at background levels (column 7). Data represent mean values ± SD of 3 separate experiments. **P < .001 versus binding of C5 or C5b6 to C18-mediated CFHR1 immobilization. (B) Formation of native CFHR1/C5 complexes in plasma was identified by immunoprecipitation. C5 antibodies were used to capture C5 complexed with CFHR1 from human plasma as shown by Western blot using polyclonal CFH antibodies (bottom panel, lane 2, arrows) and monoclonal C5 antibodies (top panel, lane 2, arrow). CFHR1 in human plasma (HP, bottom panel, lane 3) or plasma-purified CFHR1 (bottom panel, lane 4) shows similar mobilities. In addition, C5 in human plasma (top panel, lane 3) or purified C5 (top panel, lane 4) shows similar mobilities compared with the immunoprecipitated C5 protein (top panel, lane 2). Eluates derived from noncoated columns incubated with HP contain neither CFHR1 (bottom panel, lane 1) nor C5 (top panel, lane 1). The extra band in the top panel, lane 1, is considered unspecific. (C) Binding of C5 to immobilized CFHR1. CFHR1 (black line) or CFH/SCR10-13 (gray line) was immobilized to the surface of a sensor chip and C5 (50, 150, 200, 400 nM) was added in the fluid phase. (D) Binding of C5 to immobilized CFHR1 and CFH. CFHR1 (10 μg/mL) and CFH (30 μg/mL) were immobilized to a microtiter plate and incubated with increasing concentrations of C5 (1.5-50 μg/mL). Binding of C5 was detected with monoclonal C5 antibodies. Co represents reactivity of the mAb C5 to immobilized CFHR1 in the absence of C5. Representative data from 2 independent experiments are shown.