igg igm depleted human complement serum  (Pel-Freez)

Journal: Frontiers in Immunology

Article Title: An optimized Factor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae

doi: 10.3389/fimmu.2022.975676

Figure Lengend Snippet: Bactericidal activity and binding of S2477 (FH*/Fc1) and S2509 (Fc1/FH*) produced in N. benthamiana against N. gonorrhoeae in vitro . (A) Serum bactericidal activity of S2477 (FH*/Fc1) and S2509 (Fc1/FH*). The proteins were incubated (concentrations indicated in the X-axis) with N. gonorrhoeae strains FA1090, WHO X (H041) and NJ60 and 20% human complement. Survival of bacteria following incubation for 30 min relative to CFU at 0 min is expressed as a percentage on the Y-axis. The mean (SEM) of 4 separate experiments is shown. The best-fit curve used to generate the IC50 is shown. Two-way ANOVA was used to measure differences in killing across the two molecules at each concentration. **P<0.01; ***P<0.001; ****P<0.0001. (B) Binding of S2477 (FH*/Fc1) and S2509 (Fc1/FH*) to N. gonorrhoeae . The fusion proteins (concentrations indicated on the X-axis) to N. gonorrhoeae FA1090, H041 and NJ60 was measured by flow cytometry. The median fluorescence intensity (MFI) is indicated on the Y-axis. The mean (range) of two separate experiments is shown.

Article Snippet: Approximately 2000 colony forming units (CFUs) of N. gonorrhoeae were incubated with 10% human complement [IgG and IgM depleted normal human serum (Pel-Freez)] in the presence or the absence of the FH*/Fc fusion protein (concentration indicated for each experiment).

Techniques: Activity Assay, Binding Assay, Produced, In Vitro, Incubation, Bacteria, Concentration Assay, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: An optimized Factor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae

doi: 10.3389/fimmu.2022.975676

Figure Lengend Snippet: Replacing human IgG1 Fc with human IgG3 Fc improves bactericidal activity of FH*/Fc. N. gonorrhoeae FA1090 was incubated with increasing concentrations (indicated on the X-axis) of S2509 (Fc1/FH*) or S2534 (Fc3/FH*) and human complement (IgG/IgM depleted human serum) and survival at 30 min was measured (Y-axis). The mean (range) of 3 experiments is shown.

Article Snippet: Approximately 2000 colony forming units (CFUs) of N. gonorrhoeae were incubated with 10% human complement [IgG and IgM depleted normal human serum (Pel-Freez)] in the presence or the absence of the FH*/Fc fusion protein (concentration indicated for each experiment).

Techniques: Activity Assay, Incubation

Journal: Frontiers in Immunology

Article Title: An optimized Factor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae

doi: 10.3389/fimmu.2022.975676

Figure Lengend Snippet: Complement-dependent bactericidal activity of S2509 (Fc1/FH*) and S2534 (Fc3/FH*) against a panel of 50 strains of N. gonorrhoeae . Fifty strains of N. gonorrhoeae (listed on the X-axis; see Supplemental Table S1 for a description of strains) were incubated with the fusion proteins (33 µg/mL) and human complement (IgG and IgM depleted human serum; final concentration 10%). Bacterial survival at 30 min relative to 0 min is indicated as a percentage on the Y-axis. The mean (range) of two separate experiments is shown. Complement alone did not result in any killing of any strain (survival >100%) (data not shown).

Article Snippet: Approximately 2000 colony forming units (CFUs) of N. gonorrhoeae were incubated with 10% human complement [IgG and IgM depleted normal human serum (Pel-Freez)] in the presence or the absence of the FH*/Fc fusion protein (concentration indicated for each experiment).

Techniques: Activity Assay, Incubation, Concentration Assay

Journal: Frontiers in Immunology

Article Title: An optimized Factor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae

doi: 10.3389/fimmu.2022.975676

Figure Lengend Snippet: Low binding of S2534 (Fc3/FH*) limits its efficacy against PorB1A N. gonorrhoeae . (A) Complement-dependent bactericidal activity of S2534 (Fc3/FH*) against PorB1A gonococcal isolates. Ten additional PorB1A-expressing strains of N. gonorrhoeae (data with strain 252, used as a positive control for killing, has been shown in Figure 6 ) were incubated with S2534 (33 µg/mL) and human complement (IgG and IgM depleted human serum; final concentration 10%). Bacterial survival at 30 min relative to 0 min is indicated as a percentage on the Y-axis. The mean (range) of two separate experiments is shown. Complement alone did not result in any killing of any strain (survival >100%) (data not shown). (B) Binding of S2534 (Fc3/FH*; 3.3 µg/mL) to 11 PorB1A-expressing N. gonorrhoeae was measured by flow cytometry. WHO X (PorB1B) that was fully susceptible to S2534 (100% killing) was included as a comparator. The Y-axis shows the log 10 median fluorescence intensity (MFI) of fusion protein binding (mean and individual values of 2 separate experiments). The dashed grey line indicates the average value of the conjugate controls (25.7). Comparisons across groups was made with one-way ANOVA and pairwise comparisons with WHO X were made by Dunnett’s multiple comparison test. (C) Binding of C4BP to N. gonorrhoeae . The 11 PorB1A N. gonorrhoeae strains shown in panel B and PorB1B strain WHO X were incubated with 10% normal human serum (NHS) as a source of C4BP. Bound C4BP was detected with mAb 104. Binding is expressed as log 10 median fluorescence intensity (MFI) on the Y-axis (mean and individual values of two separate experiments). The dashed grey line indicates the average value of the conjugate controls (14.2). (D) The efficacy of S2534 correlates with the PorB molecule expressed. Bactericidal data from Figures 6 , 7A were compiled into the Table and analyzed with Fisher’s exact test.

Article Snippet: Approximately 2000 colony forming units (CFUs) of N. gonorrhoeae were incubated with 10% human complement [IgG and IgM depleted normal human serum (Pel-Freez)] in the presence or the absence of the FH*/Fc fusion protein (concentration indicated for each experiment).

Techniques: Binding Assay, Activity Assay, Expressing, Positive Control, Incubation, Concentration Assay, Flow Cytometry, Fluorescence, Protein Binding, Comparison