Anti fh mab

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).

Factor H (FH) binding to bacteria was performed using flow cytometry as described previously [16 (link)]. Briefly, bacteria (Ng F62 ΔlgtD) were harvested from chocolate agar plates and grown in liquid media that contained the specified concentration of the CMP-NulO as described above. Bacteria were then washed with Hanks Balanced Salt Solution (HBSS) containing 1mM Ca²⁺ and 1 mM Mg²⁺ (HBSS⁺⁺) and incubated with FH purified from human plasma (Complement Technology, Inc.; concentration specified for each experiment). Bound FH was detected using either anti-FH mAb (Quidel, catalog no. A254 (mAb 90X)) or affinity-isolated polyclonal goat anti-human FH, followed by FITC conjugated anti-mouse IgG or anti-goat IgG, respectively (Sigma); both Abs had similar performance characteristics. All reaction mixtures were carried out in HBSS⁺⁺/1% BSA in a final volume of 50 μl. Flow cytometry was performed using a FACSCalibur instrument (Becton Dickinson) and data were analyzed using FlowJo (version 7.2.5; Tree Star, Inc.).