Detecting Human FH Binding to Bacteria

Anti-FH mAb (Quidel, catalog no. A254 (mAb 90X)) or goat anti-human FH were used in flow cytometry assays to detect human FH binding to bacteria. Goat polyclonal antibodies against C3, C4 and factor B (FB) were obtained from Complement Technology, Inc (Tyler, TX). Anti-factor Bb (Quidel) added to serum at 100 μg/ml was used to block factor B function [16 (link)], thereby disabling the alternative pathway in serum bactericidal assays. Alkaline phosphatase conjugated anti-human IgG and IgM were purchased from Sigma (St. Louis, MO). mAb 3F11 (mouse IgM, kindly provided by Dr. Michael A. Apicella, University of Iowa) binds to the unsialylated HepI lacto-N-neotetraose structure; sialylation of LOS results in decreased binding of mAb 3F11 [61 (link)]. MAb 2-8C-4-1 [62 (link)] recognizes Neisserial H.8 lipoprotein and was used in whole cell ELISA assays to measure capture of bacteria on microtiter wells. FITC conjugated anti-mouse IgG and anti-goat IgG, and alkaline phosphatase conjugated anti-mouse IgM, anti-mouse IgG and anti-goat IgG were all obtained from Sigma. Neu5Gc incorporation into LNnT LOS was detected using a Neu5Gc-specific chicken polyclonal IgY Ab (1:2,000) [63 (link)] followed by FITC conjugated donkey anti-chicken IgY secondary Ab (1:200; Jackson ImmunoResearch).