Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: a Overview of the VEBIOS ER discovery cohort. 756 HPA antibodies, targeting 408 candidate proteins, were used to analyse plasma samples using affinity proteomics. Log fold changes in antibody MFI (median fluorescent intensity) signal were calculated between VTE cases ( n = 48) and controls ( n = 48) in ( b ) citrate or ( c ) EDTA anticoagulated plasma; coloured circles indicate antibodies that generated signals significantly associated with VTE in both. MFI signals generated by these antibodies for controls ( n = 48) and cases ( n = 48) in ( d ) citrate plasma and ( e ) EDTA plasma. f Immunocapture-mass spectrometry identification of protein targets of HPA059937 ( n = 3 of independent biological replicates). g Dual binder assays were developed using an anti-CFHR5 detection antibody, combined with HPA059937 (raised against SULF1), anti-CFHR5 HPA073894 or anti-CFHR5 HPA072446 as capture antibodies. Monoclonal anti-CFHR5 (MAB3845) was applied as detection antibody in the three combinations. CFHR5 levels in the citrated plasma samples were re-analysed, using the respective dual binder assays, to determine levels (MFI) in controls ( n = 48) vs . cases ( n = 48) and the correlation between the signal and those generated by the original single binder assay using HPA059937 [all p < 1E-04]. Dual binder assay using capture antibody HPA072446 with a recombinant protein standard and MAB3845 as detection antibody, was used for absolute quantification of CFHR5 in samples from: ( h ) VEBIOS ER and ( i ) VEBIOS Coagulation (controls = 135, cases = 142). CFHR5 concentration was measured in controls and cases, with associated OR (odds ratio per 1 standard deviation increase [ h and i , left]) or used to determine the correlation with C-reactive protein (CRP), or D-dimer concentration [ h and i , right panels]. All data in dot plots (1 d – i ) are represented as median value with 95% CI (confidence interval). Case and control groups was compared using a linear model adjusting for age and sex in d , e , g , h and i .***** p < 0.00001, **** p < 0.0001, *** p < 0.001, ** p < 0.01. Two-sided Spearman´s correlation analysis for h and i . For summary statistics see Supplementary data 1 [Tab_2, Panel A] . Source data are provided as a Source Data file ( b – i ).

Article Snippet: IC-MS was performed in triplicate of pooled plasma, as previously described using the HPA059937 antibody (Atlas Antibodies) or MAB3845 (R&D systems, clone:390513) and rabbit or mouse immunoglobulin G (rIgG, AB-105-C [R&D] and PMP01X [Biorad], respectively) as respective negative controls.

Techniques: Clinical Proteomics, Generated, Mass Spectrometry, Recombinant, Quantitative Proteomics, Coagulation, Concentration Assay, Standard Deviation, Control

Journal: Clinical and Experimental Immunology

Article Title: A novel fusion protein reduces kidney complement in experimental C3 glomerulopathy

doi: 10.1093/cei/uxag015

Figure Lengend Snippet: FHR5 1-9 FH 1-5 binds to C3, destabilizes C3 convertase and acts as a cofactor for factor I. Binding profiles of increasing amounts of FHR5 1-9 FH 1-5 to surface bound C3, C3b, iC3b, C3d and BSA (negative control), detected with both anti-CFHR5 (a) and anti-FH (b) antibodies. Data shown are mean values of triplicate measurements; error bars denote the standard deviation. C3a generated by convertase formation on surface-bound C3b: ELISA plates were coated with purified C3b. Surface C3 convertase was formed by addition of purified factor B (FB), factor D (FD), and properdin (P). Then increasing amounts of either FH (c) or the FHR5 1-9 FH 1-5 (d) were added followed by addition of C3 to enable C3a generation. To investigate factor I (FI) co-factor activity, FI along with increasing amounts of either FH (e) or the FHR5 1-9 FH 1-5 (f) were added prior to the convertase formation step. NC—negative control, no FB, FD, or P added. PC—positive control, no FH, or FHR5 1-9 FH 1-5 added. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test; **** P < 0.0001. Fluid phase co-factor assay products were visualized by western blotting (g). Cofactor activity was indicated by the cleavage of C3b as determined by the presence of C3 α-chain fragments (arrow). Negative controls: C3, C3b, C3b with FH, C3b with FI, and FHR5 1-9 FH 1-5 alone. Positive control: C3b incubated with FH and FI.

Article Snippet: After incubation and washing, bound FHR5 1-9 FH 1-5 was detected using goat polyclonal anti-CFHR5 antibody (R&D systems), followed by mouse anti-goat/sheep IgG-HRP (Sigma) and finally TMB substrate (BD).

Techniques: Binding Assay, Negative Control, Standard Deviation, Generated, Enzyme-linked Immunosorbent Assay, Purification, Activity Assay, Positive Control, Western Blot, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gain-of-function factor H–related 5 protein impairs glomerular complement regulation resulting in kidney damage

doi: 10.1073/pnas.2022722118

Figure Lengend Snippet: FHR5mut triggers glomerular C3 deposition in hFH-FHR5 mice. ( A ) Representative glomerular images and ( B ) staining scores for C3b/iC3b/C3c, C3d, FHR5/FHR5mut, FH, IgG, and properdin in male hFH-FHR5 mice treated with either AAV-GFP ( n = 5, black dots) or AAV-FHR5mut ( n = 6, red dots). Bars denote the median, and whiskers denote the interquartile range. * P = 0.0022; values are derived from Mann–Whitney U test. Plasma FHR5/FHR5mut ( C ), C3 ( D ), and FH ( E ) levels after administration of either AAV-FHR5mut or AAV-GFP to hFH-FHR5 male mice. Bars denote the mean, and whiskers denote the SD. P values are derived from two-way ANOVA with Bonferroni multiple comparisons test. ( F ) Representative glomerular images of C3b/iC3b/C3c, C3d, and FHR5mut in male hFH-FHR5mut mice expressing one copy of the transgene on the background of either heterozygous (delFH-FHRhet.hFH-FHR5mut) or homozygous (hFH-FHR5mut) deficiency of the mouse FH-FHR locus. Controls are male delFH-FHRhet.hFH-FHR5 and hFH-FHR5 animals.

Article Snippet: IF antibodies were as follows: C3b/iC3b/C3c, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse C3 (MP Biomedicals, 855500); C3d, biotinylated goat anti-mouse C3d (R&D system, BAF2655); mouse FH and mouse FHR proteins, polyclonal goat anti-hFH (Quidel), A312; hFH, polyclonal goat anti-hFH (Quidel, A312); FHR5/FHR5mut, rabbit monoclonal anti-human CFHR5 antibody (Abnova, H00081494-D01P); mouse properdin, FITC-conjugated goat anti-human properdin (Caltag Madsystems, GAHu/PPD/FITC); and mouse IgG, FITC-conjugated polyclonal goat anti-mouse IgG Fc γ-chain–specific antibody (Sigma-Aldrich, F5387).

Techniques: Staining, Derivative Assay, MANN-WHITNEY, Clinical Proteomics, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Gain-of-function factor H–related 5 protein impairs glomerular complement regulation resulting in kidney damage

doi: 10.1073/pnas.2022722118

Figure Lengend Snippet: HDM-FH ameliorates glomerular C3 in hFH-FHR5mut mice. ( A ) Representative glomerular images and ( B ) staining scores for C3b/iC3b/C3c, C3d, FHR5mut, FH, IgG, and properdin in male hFH-FHR5mut mice treated with either AAV-GFP ( n = 5, black dots) or AAV-HDM-FH ( n = 7). Bars denote the median, and whiskers denote the interquartile range. * P = 0.0013, derived from Mann–Whitney U test. Plasma hFH/HDM-FH ( C ), C3 ( D ), and FHR5mut ( E ) levels after administration of either AAV-GFP or AAV-HDM-FH to hFH-FHR5mut male mice. Bars denote the mean, and whiskers denote the SD. P values are derived from two-way ANOVA with Bonferroni multiple comparisons test.

Article Snippet: IF antibodies were as follows: C3b/iC3b/C3c, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse C3 (MP Biomedicals, 855500); C3d, biotinylated goat anti-mouse C3d (R&D system, BAF2655); mouse FH and mouse FHR proteins, polyclonal goat anti-hFH (Quidel), A312; hFH, polyclonal goat anti-hFH (Quidel, A312); FHR5/FHR5mut, rabbit monoclonal anti-human CFHR5 antibody (Abnova, H00081494-D01P); mouse properdin, FITC-conjugated goat anti-human properdin (Caltag Madsystems, GAHu/PPD/FITC); and mouse IgG, FITC-conjugated polyclonal goat anti-mouse IgG Fc γ-chain–specific antibody (Sigma-Aldrich, F5387).

Techniques: Staining, Derivative Assay, MANN-WHITNEY, Clinical Proteomics