soluble cr1 (scr1)  (Cell Sciences Inc)

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Kinetic and equilibrium dissociation constants for the binding of C1q to immobilized soluble CR1 (sCR1) and CR1 CCP22-30.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Localization of the C1q binding sites in CR1 CCP22-30 using deletion fragments. (A) Schematic view of the different fragments of CR1 CCP22-30. Potential N-glycosylations are represented by open circles ○. (B) Interaction of C1q with immobilized CR1 CCP22-30 fragments analyzed by ELISA. C1q (10 µg/ml in PBS) was added to microtiter plate coated with 3.4 pmol of each CCP22-30 fragment as described in Section “ .” After 1.5 h incubation at room temperature, bound C1q was detected with rabbit antibodies recognizing C1q and HRP secondary antibodies. Data are presented as the mean ± SE of four individual experiments. (**), Student’s t -test values p < 0.01 of C1q binding to each fragment compared to CCP22-30.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Complement receptor type 1 (CR1) variants produced to study the CCP24-25 module pair interaction properties. (A) Schematic view of CCP24-25 variants produced in eukaryotic cells. Potential N-glycosylations are represented by open circles ○. (B) SDS-PAGE analysis of 4 µg of the CR1variants under reducing conditions. The positions of the molecular weight markers (expressed in kilodaltons) are indicated. A full scan of the original gel is provided in Figure S1 in Supplementary Material .

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Produced, SDS Page, Molecular Weight

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Kinetic analysis of the interaction of C1q and mannose-binding lectin (MBL) with CR1 CCP24-25. C1q (A) and MBL (B) , at indicated concentrations, were injected in 50 mM triethanolamine-HCl (TEA), 145 mM NaCl, 0.05% surfactant P20, pH 7.4, on immobilized CR1 CCP24-25 (1,000 RU). The buffer was supplemented with 3 mM EDTA for MBL binding. Fits are shown as dotted lines and were obtained by global fitting of the data using a 1:1 Langmuir-binding model. The kinetic constants obtained are framed on the top of each sensorgramm.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, Injection

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Interaction of C1q and mannose-binding lectin (MBL) with immobilized CR1 CCP22-30 and CR1 ΔCCP24-25. The binding curves were obtained in single cycle mode by injecting increasing concentrations of C1q (A) or MBL (B) on immobilized CR1 CCP22-30 (3,000 RU) or CR1 ΔCCP24-25 (2,200 RU). C1q (0.25, 0.5, 1, 2, and 4 nM) was injected at 20 µl/min for 180 s in 50 mM triethanolamine-HCl (TEA), 150 mM NaCl, 1 mM CaCl 2 , 0.05% surfractant P20, pH 7.4. MBL (1, 2, 4, 8, and 16 nM) was injected in the same conditions except that the buffer contained 3 mM EDTA instead of CaCl 2 .

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, Injection

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Elongated shape of CCP24-25, associated to a possible interpretative model. (A) Small-angle X-ray scattering (SAXS) pair distance distribution, (B) SAXS dimensionless Kratky plot, which shows elongation and remaining flexibility. (C) Fit of the model [shown in panels (D,E) ] to the experimental data. (D) Top and (E) side views of an ab initio envelope computed with GASBOR. A Coral-derived model of CCP24-25 is shown inside to illustrate how the shape corresponds to two CCP modules. This interpretative model is used to illustrate the positions on each module of glycosylation sites (green), rs 4844609 SNP (magenta), Knops groups variants (cyan), acidic clusters (red) initially suggested as potential MBL binding sites, and the other acidic residues (gray and black). D1529 is the homologous counterpart of D1076 in CR1 CCP17 (black). The two flexible carbohydrates included in the model are only illustrated in the side view.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Derivative Assay, Glycoproteomics, Binding Assay

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Localization of the binding site of soluble CR1 on C1q. (A) Comparative binding of C1q and its CLF and GR on complement receptor type 1 (CR1) CCP22-30 analyzed by surface plasmon resonance (SPR). The CR1 CCP22-30 fragment was immobilized on CM5 sensor chips (4,500 RU) and 2 nM of C1q or CLF and 12 nM of GR were injected over the surfaces as described in Section “ .” (B) MBL-associated serine protease (MASP)-3 competition of the binding of C1q to CR1 CCP22-30 analyzed by SPR. C1q (2 nM) was incubated at room temperature for 20 min in the absence or presence of recombinant MASP-3 at indicated molar ratios and injected over immobilized CR1 CCP22-30 (4,500 RU). No binding was observed when MASP-3 alone (20 nM) was injected.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, SPR Assay, Injection, Incubation, Recombinant

Journal:

Article Title: Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

doi: 10.4049/jimmunol.0902810

Figure Lengend Snippet: Binding of sCR1 to nonactivated and TRAP-activated platelets exposed to native C3. The background level of sCR1 binding to platelets without the presence of C3 has been subtracted from the levels obtained in the presence of C3 for each individual experiment. The data are shown as means ± SEM (n = 7; *p < 0.05).

Article Snippet: Soluble CR1 (sCR1) BRL55730 was a kind gift from Dr. Henry Marsh (GlaxoSmithKline, Research Triangle Park, NC).

Techniques: Binding Assay

Journal: PLoS Pathogens

Article Title: Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

doi: 10.1371/journal.ppat.1000968

Figure Lengend Snippet: (A) Binding of chicken anti-human CR1 Fab is abolished by treatment of erythrocytes with trypsin but not with neuraminidase. (B) Binding of chicken anti-CR1 Fab to human red cells is abolished by incubation with soluble CR1 (sCR1) but not by bovine serum albumin. (C) PAGE of chicken anti-CR1 immunoprecipitates, sCR1 and red cell lysates run on Novex Nupage Bis-tris 4–12% acrylamide gel under reducing conditions and stained with silver. Lane 1: Immunoprecipitate with chicken anti-CR1 from intact red cell lysate. Lane 2: Immunoprecipitate with chicken anti-CR1 from trypsin-treated red cell lysate. Lane 3: Immunoprecipitate with control chicken IgY from intact red cell lysate. Lane 4: Immunoprecipitate with chicken IgY from trypsin-treated red cell lysate. Lane 5: 0.01 µg of purified sCR1. Lane 6: 1 µl of intact red cell lysate used for immunoprecipitation. Lane 7: 1 µl of trypsin-treated red cell lysate used for immunoprecipitation.

Article Snippet: For blocking with soluble CR1 (sCR1) (AVANT Immunotherapeutics, Inc., Needham, MA, USA), bovine serum albumin, fetuin, and α-2-macroglobulin (Sigma-Aldrich) were used as negative control proteins depending on their availability.

Techniques: Binding Assay, Incubation, Acrylamide Gel Assay, Staining, Control, Purification, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

doi: 10.1371/journal.ppat.1000968

Figure Lengend Snippet: (A) sCR1 blocked invasion of neuraminidase-treated erythrocytes in a dose-dependent manner while bovine serum albumin (BSA) and α-2-macroglobulin had no effect. (B) No protein had an effect on the invasion of untreated erythrocytes. (C) Chicken anti-human CR1 Fab blocked invasion of neuraminidase-treated erythrocytes in a dose-dependent manner while purified chicken IgY Fab control had no effect. (D) No antibody had effect on the inhibition of intact red cells. (C–D) The concentration of antibody stocks was 80 µg/ml. Summary of three experiments. (E) Inhibition of P. falciparum 7G8 invasion using anti-CR1 monoclonal antibodies. Numbers above bars are P values for the comparison with the no antibody (No Ab) control using Dunnett's pairwise multiple comparison t-test, taking into account matching by experiment date. An IgG 1 irrelevant monoclonal (R&D Systems, Minneapolis, MN, USA) and anti-CD55 monoclonal (clone NaM16-4D3) (4D3) (Santa Cruz Biotechnology, Inc.) were used as negative controls. E11, To5, J3D3, and J3B11 are anti-CR1 monoclonal antibodies. All monoclonal antibodies were used at 1 µg/ml except in two experiments where every monoclonal was used at 15 µg/ml. Bars represent means ± standard deviations. Invasion rates in untreated controls (no antibody) ranged from 2% to 14%.

Article Snippet: For blocking with soluble CR1 (sCR1) (AVANT Immunotherapeutics, Inc., Needham, MA, USA), bovine serum albumin, fetuin, and α-2-macroglobulin (Sigma-Aldrich) were used as negative control proteins depending on their availability.

Techniques: Purification, Control, Inhibition, Concentration Assay, Bioprocessing, Comparison

Journal: PLoS Pathogens

Article Title: Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

doi: 10.1371/journal.ppat.1000968

Figure Lengend Snippet: HB3 invasion of neuraminidase-treated cells was least affected by monoclonal and polyclonal antibodies against CR1. Monoclonal antibodies were used at 4 µg/ml. Fab fragments were used at 8 µg/ml and purified proteins were used at 50 µg/ml. Numbers above bars are P values for the comparison with the no-inhibitor control using Dunnett's pairwise multiple comparison t test, taking into account matching by experiment date. Bars represent means ± standard deviations. Alpha-2-mac = α-2-macroglobulin. Invasion rates for untreated erythrocytes with no inhibitor ranged from 3% to 37%.

Article Snippet: For blocking with soluble CR1 (sCR1) (AVANT Immunotherapeutics, Inc., Needham, MA, USA), bovine serum albumin, fetuin, and α-2-macroglobulin (Sigma-Aldrich) were used as negative control proteins depending on their availability.

Techniques: Bioprocessing, Purification, Comparison, Control

Journal: PLoS Pathogens

Article Title: Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

doi: 10.1371/journal.ppat.1000968

Figure Lengend Snippet: CR1 shows a characteristic speckled pattern with aggregation around the merozoite. All negative control antibodies showed no staining. CR1 and glycophorin A were stained with specific antibodies followed by fluorochrome-conjugated secondary antibodies against glycophorin (red) and CR1 (green). Merozoites (blue) were stained with the nucleic acid specific stain (Hoechst 33342). DIC = differential interference contrast.

Article Snippet: For blocking with soluble CR1 (sCR1) (AVANT Immunotherapeutics, Inc., Needham, MA, USA), bovine serum albumin, fetuin, and α-2-macroglobulin (Sigma-Aldrich) were used as negative control proteins depending on their availability.

Techniques: Negative Control, Staining

Journal: PLoS Pathogens

Article Title: Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

doi: 10.1371/journal.ppat.1000968

Figure Lengend Snippet: (A) Protein-coated microspheres were incubated overnight with purified late trophozoites and schizonts. Binding was detected by % of spheres positive for Hoechst staining using flow cytometry. Bars represent means ± standard deviations. The figure represents a summary of 2–3 experiments except for the rabbit anti-BSA which was included in one experiment. (B) Fluorescence image of sCR1-coated microspheres showing attached merozoites (blue) and surface sCR1 (green). (C) Differential interference contrast image of microspheres in B.

Article Snippet: For blocking with soluble CR1 (sCR1) (AVANT Immunotherapeutics, Inc., Needham, MA, USA), bovine serum albumin, fetuin, and α-2-macroglobulin (Sigma-Aldrich) were used as negative control proteins depending on their availability.

Techniques: Incubation, Purification, Binding Assay, Staining, Flow Cytometry, Fluorescence

Journal: PLoS Pathogens

Article Title: Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

doi: 10.1371/journal.ppat.1000968

Figure Lengend Snippet: Erythrocytes from 27 healthy donors were used. To control for day-to-day variation the percent invasion as well as the CR1 MFI were normalized to an erythrocyte standard sample that was used in every experiment. The CR1 MFI was re-measured once in 4 samples with extreme values. In 19 donors the invasion assay was repeated 1–3 times. When more than one measurement was done, the mean was used for normalization.

Article Snippet: For blocking with soluble CR1 (sCR1) (AVANT Immunotherapeutics, Inc., Needham, MA, USA), bovine serum albumin, fetuin, and α-2-macroglobulin (Sigma-Aldrich) were used as negative control proteins depending on their availability.

Techniques: Control, Invasion Assay

Journal: PLoS Pathogens

Article Title: Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

doi: 10.1371/journal.ppat.1000968

Figure Lengend Snippet: (A) 7G8 invaded CR1 transgenic mouse erythrocytes preferentially in a mixed culture of wild-type and CR1 transgenic mouse erythrocytes incubated overnight with or without schizonts and analysed by flow cytometry. Upper panels show dot plots of Hoechst negative (uninfected) and positive (infected) erythrocytes. Numbers in parenthesis represent the percent parasitemia (%P). Numbers without parenthesis in each quadrant represent the percent cells from the total population. The lower panels show CR1 fluorescence histograms and the demarcation between CR1 positive (rectangle) and negative erythrocytes. (B) Effect of polyclonal and monoclonal antibodies on the invasion of wild-type and CR1 transgenic mouse erythrocytes by 7G8. Only significant P values are shown. *P is for the comparison between wild-type and CR1 transgenic red cells with no antibody. #P is for the comparison between antibody and no antibody. Invasion in wild-type erythrocytes ranged from 0.2% to 4.5%. (C) Comparison of invasion of wild-type and CR1 transgenic mouse erythrocytes between 7G8 and Dd2. Numbers above bars represent P values for the comparison to the wild-type untreated control. Invasion was measured by flow cytometry and ranged from 0.45% to 5.5% in wild-type untreated erythrocytes. All bars represent means ± standard deviations.

Article Snippet: For blocking with soluble CR1 (sCR1) (AVANT Immunotherapeutics, Inc., Needham, MA, USA), bovine serum albumin, fetuin, and α-2-macroglobulin (Sigma-Aldrich) were used as negative control proteins depending on their availability.

Techniques: Transgenic Assay, Incubation, Flow Cytometry, Infection, Fluorescence, Bioprocessing, Comparison, Control