To study the effects of the FH18-20/Fc proteins without confounding by natural anti-gonococcal antibodies present in NHS, we depleted IgG and IgM from freshly collected human serum, as described previously (33 (link)). Briefly, EDTA (final concentration 10 mM) and NaCl (final concentration 1 M) were added to freshly prepared human serum and treated sera was passed first over anti-human IgM agarose (Sigma), followed by passage through protein G-Sepharose; both columns were equilibrated in PBS containing 10 mM EDTA and 1 M NaCl. NaCl was added to minimize loss of C1q during passage of serum through the anti-human IgM column. The flow-through was collected, spin concentrated and dialyzed against PBS/0.1 mM EDTA to its original volume using a 10-kDa cutoff Amicon Ultra-15 centrifugal filter device (Millipore, Bedford, MA), sterilized by passage through a 0.22-μm filter (Millipore), aliquoted and stored at −70 °C. Hemolytic activity was confirmed using a total complement hemolytic plate assay (The Binding Site Inc., Birmingham, U.K). Depletion of IgG and IgM was confirmed by dot-blot assays using alkaline phosphatase conjugated goat anti-human IgG and goat anti-human IgM, respectively. In some experiments, complement activity of serum was destroyed by heating serum at 56°C for 1 h.