goat polyclonal anti cfhr5 antibody  (R&D Systems)

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: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: The primers used to generate the FHR5 fragments used in this study.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: Schematic drawing of FH, FHR1, FHR5 and the recombinant FHR5 fragments used in this study. FH, FHR1 and FHR5 are composed of individually folding globular domains called complement control protein domains (CCPs) or also known as short consensus repeats (SCRs). CCPs 1-4 of FH mediate complement regulatory activity while the CCPs 7 and 18-20 are responsible for binding different ligands and surface recognition. The CCPs of FHR1 and FHR5 share high amino acid sequence identity with the corresponding domains of FH, indicated by numbers above. The C-terminal part is well conserved indicating similar or identical ligand and surface binding between FHR1, FHR5 and FH. FHR proteins lack the N-terminal regulatory domains CCPs 1-4 of FH. CCPs 1-2 of FHR1 and FHR5 are very similar to each other and responsible for formation of homo and heterodimers.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Recombinant, Control, Activity Assay, Binding Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: Binding of FHR1 and FHR5 to ECM components. FHR1 (A) and FHR5 (B) binding to ECM ligands was analyzed by protein microarray. ECM proteins, gelatin and MaxGel were printed onto nitrocellulose-covered slides in triplicates. Air-dried slides were washed and blocked with 4% BSA, then incubated with FHR1 or FHR5 in increasing concentrations. Bound proteins were detected with polyclonal goat anti-human FH or polyclonal goat anti-human FHR5 Ab and Alexa-647 labeled goat-IgG. A signal higher than the one obtained for the negative control protein gelatin was defined as the threshold for binding. Data are representative of two experiments.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Binding Assay, Microarray, Incubation, Labeling, Negative Control

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: FHR5 binds through its middle part, CCPs 3-7, and FHR1 binds via its C-terminal domain to ECM proteins. In ELISA 10 µg/ml of gelatin, laminin, PRELP (A) , osteoadherin and fibromodulin (B) were immobilized in microplate wells and after blocking, incubated with 200 nM (A) or 100 nM (B) FHR5, CCPs 1-4, CCPs 3-7 and CCPs 8-9. Binding of FHR5 and its fragments was measured with goat anti-human FHR5 Ab. Data are means ± SD derived from three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 one-way ANOVA, compared to the negative control, gelatin. (C) Laminin, fibromodulin, osteoadherin and PRELP were immobilized (10 µg/ml) in microplate wells. After blocking, FHR1 was added in increasing concentrations. Binding of FHR1 was detected with polyclonal goat anti-human FH and the corresponding secondary antibody. Data are means ± SD derived from three independent experiments. *p <0.05, **p <0.01, ***p <0.001 two-way ANOVA, compared to the negative control, HSA; ns, not significant. (D) Immobilized ECM proteins were incubated with FHR1 (5 µg/ml) in the presence or absence of the monoclonal Ab C18, which recognizes the CCP 5 domain of FHR1. We used monoclonal Ab A255 as a negative control. Bound FHR1 was detected as described earlier. Data are means ± SD derived from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 two-way ANOVA.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation, Binding Assay, Derivative Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: FHR1 and FHR5 compete with FH for binding to several ECM components, and FHR5 competitively inhibits the cofactor activity of FH. ECM proteins were printed on slides and incubated with 25 µg/ml FH together with increasing concentrations of FHR1 (A) or FHR5 (B) . Binding of FH was measured by monoclonal mouse anti-FH (A254) that does not recognize FHR1 or FHR5 and Alexa546-conjugated goat anti-mouse IgG. Data are representative of two experiments. (C) Competition between FHR5 and FH was also measured in ELISA. Laminin, fibromodulin, osteoadherin and PRELP (10 µg/ml of each) were immobilized and incubated with 50 µg/ml FH with or without 20 µg/ml FHR5. FH binding to ECM proteins was detected as described earlier. The values were normalized for FH binding (100%) and show means ± SD derived from three independent experiments. *p <0.05, **p <0.01, ***p <0.001, one-way ANOVA; ns, not significant. (D) Laminin (10 µg/ml) was immobilized and after blocking, the wells were incubated with 100 µg/ml FH with or without 20 µg/ml FHR5. Then, 140 nM C3b and 220 nM FI were added to the wells for one hour at 37°C. After incubation, supernatants were analyzed on 7.5% SDS-PAGE and Western blot. The blot was developed by using HRP-conjugated anti-human C3 Ab that recognizes C3b and its fragments except for C3d. The molecular mass marker is indicated on the left, and the C3b chains and the C3b α’-chain cleavage fragments are indicated on the right. Results are representative of three experiments.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Binding Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay, Blocking Assay, SDS Page, Western Blot, Marker

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: Binding of FHR5 and its fragments to C3b, iC3b, C3c and C3d. (A) FHR5, CCPs 1-4, CCPs 3-7, CCPs 8-9 and HSA were immobilized at 5 µg/ml in microplate wells. After blocking, 20 µg/ml C3b, iC3b and C3c were added. Bound proteins were detected using HRP-conjugated anti-human C3 Ab. (B) C3d binding to FHR5 also was measured by ELISA. FHR5 and its fragments were coated and incubated with 10 µg/ml C3d. Binding of C3d was measured with an anti-human C3d Ab and the corresponding secondary Ab. (C) In reverse setting, C3b was coated (20 µg/ml) and, after blocking, microplate wells were incubated with 20 µg/ml FHR5, CCPs 1-4, CCPs 3-7 and CCPs 8-9. HSA was used as a control protein. Binding of FHR5 and its fragments was detected by a polyclonal goat anti-FHR5 and HRP-conjugated secondary Ab. (D) Dose-dependent binding of C3b to FHR5 and its fragments was measured as shown in (A) ; C3b was added in increasing concentrations. Data are means ± SD derived from five (A–C) or two (D) independent experiments. *p < 0.05, ***p < 0.001, one-way ANOVA. (E) Binding of C3, C3(H 2 O) and C3b to immobilized FHR5 and its fragments was measured by ELISA using HRP-conjugated polyclonal anti-C3 antibody. Data are means + SD derived from four experiments. **p < 0.01, ***p < 0.001, one-way ANOVA, compared to the negative control protein alpha-1-antitrypsin (a-1-AT).

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Incubation, Control, Protein Binding, Derivative Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: Analysis of properdin binding to FHRs. (A) The five FHR proteins, C3b as positive control protein and HSA as negative control protein were immobilized and binding of purified properdin (P) and the isolated properdin dimers, trimers and tetramers (P2, P3 and P4, respectively) was measured by ELISA using anti-properdin antibody. (B) Properdin binding to the FHR5 deletion mutants CCPs 1-4, 3-7 and 8-9 was measured as in (A) . Data are means + SD from three experiments. ***p <0.001, one-way ANOVA, compared to the negative control protein HSA.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Binding Assay, Positive Control, Negative Control, Purification, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: FHR1 increases C3 fragment deposition on ECM proteins. Immobilized ECM proteins were incubated with 10% NHS with or without FHR1 added in increasing concentrations (10 µg/ml, 20 µg/ml, 40 µg/ml) diluted in 5 mM Mg 2+ -EGTA. C3 fragment deposition (A) was measured with HRP-conjugated polyclonal anti-human C3 Ab and FB binding (B) was measured by goat anti-human FB and the corresponding secondary Ab. Data are means ± SD derived from four independent experiments. *p <0.05, ***p < 0.001, two-way ANOVA; ns, not significant.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Incubation, Binding Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: FHR5 causes enhanced C3 fragment deposition and supports alternative pathway activation when bound to ECM proteins. Immobilized ECM proteins were incubated with 10% NHS in the presence or absence of 10 µg/ml FHR5, supplemented with 5 mM Mg 2+ -EGTA which allows only alternative pathway activation. Binding of C3 fragments and factor B was detected with HRP-conjugated anti-human C3 Ab (A) and goat anti-human FB Ab (B) . Data are means ± SD derived from three experiments. **p < 0.01, ***p < 0.001, one-way ANOVA.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Activation Assay, Incubation, Binding Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: FHR5 increases C5b-9 deposition on ECM ligands. ECM proteins were printed onto nitrocellulose-covered slides and after blocking, proteins were incubated with normal human serum (NHS) in the presence or absence of recombinant FHR1 (50 µg/ml) or FHR5 (20 µg/ml) for 1 hour at 37°C. Bound proteins were detected with polyclonal anti-C3 (A) or monoclonal anti-sC5b-9 (B) and with the corresponding secondary Ab. Data are representative of two independent experiments.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Blocking Assay, Incubation, Recombinant

Journal: Frontiers in Immunology

Article Title: Complement Factor H-Related Proteins FHR1 and FHR5 Interact With Extracellular Matrix Ligands, Reduce Factor H Regulatory Activity and Enhance Complement Activation

doi: 10.3389/fimmu.2022.845953

Figure Lengend Snippet: Schematic overview of the interaction between FH/FHRs and the ECM. FHR1 and FHR5 can bind to ECM components and competitively inhibit the binding of FH, thus reducing the complement regulatory activity of FH. The ECM-bound FHR1 and FHR5 may also directly enhance alternative pathway activation by binding C3(H 2 O) and C3b and allowing formation of C3 convertase.

Article Snippet: Polyclonal goat anti-human FHR5 was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Binding Assay, Activity Assay, Activation Assay