Journal: Frontiers in Immunology

Article Title: Toxoplasma gondii Recruits Factor H and C4b-Binding Protein to Mediate Resistance to Serum Killing and Promote Parasite Persistence in vivo

doi: 10.3389/fimmu.2019.03105

Figure Lengend Snippet: Toxoplasma gondii recruits AP regulator Factor H and CP/LP regulator C4b-binding protein to the parasite surface. 1 × 10 6 Type II ME49 parasites were incubated in 10% NHS for 0–60 min at 37°C. Western blots (left panels) of C4BP (A) (rabbit α-human C4BP, AssayPro 1:500) and FH (B) (goat α-human Factor H, CompTech 1:20,000) binding. Serum or purified protein was used as a positive control and heat inactivated serum (hiNHS) was used as a negative control. Blots were stripped and re-probed with anti-SRS29B (SAG1) for loading control. Images are from one representative of three independent experiments with similar results. Right panels (A,B) represent flow cytometric assays of C4BP (A) and FH (B) binding to the parasite surface for 0–60 min. Heat inactivated NHS (hiNHS) serum was used as a negative control. Flow cytometry data are shown as mean ± SEM from three independently performed experiments.

Article Snippet: The following antibody dilutions were used: rabbit anit-SRS29B (formerly SAG1, 30 kDa) 1:5,000, rabbit anti-C4BP (alpha chain, 70 kDa) (AssayPro) 1:1,000, goat anti-C3 (C3, C3b, iC3b) and goat anti-Factor H (full length protein, 155 kDa) antibodies (CompTech) were both used at 1:20,0000, anti-rabbit HRP 1:10,000 (Sigma), and anti-goat HRP 1:5,000 (Santa Cruz Biotechnology, Inc.).

Techniques: Binding Assay, Incubation, Western Blot, Purification, Positive Control, Negative Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Toxoplasma gondii Recruits Factor H and C4b-Binding Protein to Mediate Resistance to Serum Killing and Promote Parasite Persistence in vivo

doi: 10.3389/fimmu.2019.03105

Figure Lengend Snippet: Factor H and C4b-binding protein contribute to serum resistance. Factor H (FH) and C4b-binding protein (C4BP) were blocked by pre-incubating 10% NHS with 1:100 or 1:400 dilution of goat α-human FH (CompTech) or rabbit α-human C4BP (AssayPro) for 1 h on ice before adding to 1 × 10 6 parasites and incubating for 60 min at 37°C. Flow cytometric analysis of C5b-9 formation (A) and parasite viability (B) after 60' in 10% NHS blocked with 1:100 or 1:400 of α-C4BP (gray bars) α-FH (open bars) antibodies. 10% heat inactivated serum (hiNHS) was used a negative control. Flow cytometry data are shown as mean ± SEM from three independently performed experiments. Significant differences between the compared groups was determined using multiple Student's t test with Holm-Sidak correction for multiple comparisons, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibody dilutions were used: rabbit anit-SRS29B (formerly SAG1, 30 kDa) 1:5,000, rabbit anti-C4BP (alpha chain, 70 kDa) (AssayPro) 1:1,000, goat anti-C3 (C3, C3b, iC3b) and goat anti-Factor H (full length protein, 155 kDa) antibodies (CompTech) were both used at 1:20,0000, anti-rabbit HRP 1:10,000 (Sigma), and anti-goat HRP 1:5,000 (Santa Cruz Biotechnology, Inc.).

Techniques: Binding Assay, Negative Control, Flow Cytometry

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Exploring the function of factor XIII free B subunit: Interactions with complement factors and a novel approach to identify potential binding partners

doi: 10.1002/rth2.12766

Figure Lengend Snippet: Cofactor activity in the degradation of complement C3b and C4b. C3b and C4b were incubated with FI in presence of cofactors FH and C4BP, respectively, and/or free FXIII‐B. (A) The individual proteins C3b, FI, and FXIII‐B are shown, followed by a time course of the incubation with FXIII‐B. The last lane shows as positive control the degradation of C3b by FI in the presence of FH and the degradation products are indicated with arrows. (B) The individual proteins C4b, FI, and FXIII‐B are shown, followed by a time course of the incubation with FXIII‐B. The last lane shows as positive control the degradation of C4b by FI in the presence of C4BP and the degradation products are indicated with arrows. (C) C3b was incubated with FI and FH over 15 min, and in every second sample FXIII‐B was added. (D) C4b was incubated with FI and C4BP over 15 min, and in every second sample FXIII‐B was added. C4BP, C4b‐binding protein; FH, factor H; FI, factor I; FXIII‐B, factor XIII‐B

Article Snippet: Plasma‐purified C3b, C4b, FH, C4BP, and factor I (FI) were from Complement Technology Inc. We used polyclonal goat anti‐human C3, goat anti‐human C4, goat anti‐human FH antibodies (Complement Technology Inc., Tyler, TX, USA), rabbit anti–FXIII‐B polyclonal antibody (Calbiochem, Merck, San Diego, CA, USA), a monoclonal murine anti‐human C4BP antibody (Quidel, San Diego, CA, USA), and secondary peroxidase‐labeled anti‐goat IgG, anti‐rabbit IgG, anti‐mouse IgG, and goat anti‐rabbit alkaline peroxidase–labeled antibody (Sigma‐Aldrich/Merck).

Techniques: Activity Assay, Incubation, Positive Control, Binding Assay

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Exploring the function of factor XIII free B subunit: Interactions with complement factors and a novel approach to identify potential binding partners

doi: 10.1002/rth2.12766

Figure Lengend Snippet: ELISA‐based binding assays. (A) Dose‐dependent binding of FH and free FXIII‐B to C3b coated onto a microplate. (B) Dose‐dependent binding of C4BP and free FXIII‐B to C4b coated onto a microplate. (C) Dose‐dependent binding of C3b to FH, FXIII‐B and BSA coated onto a microplate. (D) Dose‐dependent binding of C4b to C4BP, FXIII‐B, and BSA coated onto a microplate. BSA, bovine serum albumin; C4BP, C4b‐binding protein; FH, factor H; FXIII‐B, factor XIII‐B

Article Snippet: Plasma‐purified C3b, C4b, FH, C4BP, and factor I (FI) were from Complement Technology Inc. We used polyclonal goat anti‐human C3, goat anti‐human C4, goat anti‐human FH antibodies (Complement Technology Inc., Tyler, TX, USA), rabbit anti–FXIII‐B polyclonal antibody (Calbiochem, Merck, San Diego, CA, USA), a monoclonal murine anti‐human C4BP antibody (Quidel, San Diego, CA, USA), and secondary peroxidase‐labeled anti‐goat IgG, anti‐rabbit IgG, anti‐mouse IgG, and goat anti‐rabbit alkaline peroxidase–labeled antibody (Sigma‐Aldrich/Merck).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: Recombinant constructs and purified proteins. (A) Diagram of the domain distribution of MAP-1, C4BP α-chain and the recombinant proteins. (B) Instant blue stain of purified MAP-1:C4BP 1−5 , C4BP 1−5 :MAP-1, and C4BP 1−5 via affinity chromatography. (C) The identity of the protein bands was confirmed by Western blotting using anti-MAP-1 mAb and rMAP-1 as positive control.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Recombinant, Construct, Purification, Staining, Affinity Chromatography, Western Blot, Positive Control

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: Gel filtration chromatography of chimeric proteins. (A) Gel filtration profile of MAP-1:C4BP 1−5 (M:C) and C4BP 1−5 :MAP-1 (C:M) under physiological calcium conditions or 10 mM EDTA. The identity of the gel filtration profiles was confirmed by analyzing the elution fractions of M:C (B) or C:M (C) by sandwich ELISA using anti-C4BP as capture antibody and anti-MASP-1/-3/MAP-1 as detection. Relative abundance of the chimeric proteins in the elution fractions following gel filtration under calcium conditions or EDTA is expressed as OD. mAU, milli absorption units.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Filtration, Chromatography, Sandwich ELISA

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: MAP-1:C4BP 1−5 (M:C), C4BP 1−5 :MAP-1 (C:M) and MAP-1 binding to MBL (A) and CL-11 (B) . rMBL and rCL-11 were immobilized onto mannan-coated plates. Recombinant proteins were applied in a two-fold dilution in the presence of calcium or EDTA. Binding was determined using an anti-MAP-1 mAb. Connecting lines display nonlinear fitting using the equation specific binding with hill slope. Results are representative of three independent experiments and error bars represent minimum and maximum values of triplicate measurements.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Binding Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: Chimeric and rMAP-1 complement inhibition after pre-incubation on rMBL/mannan. Serial dilutions of MAP-1:C4BP 1−5 (M:C), C4BP 1−5 :MAP-1 (C:M), and rMAP-1 were allowed to form complexes with mannan-bound rMBL prior to addition of MBL-defect serum as a source of complement. Detection of C4 (A) , C3 (B) , and TCC (C) deposition was quantified using anti-C4, anti-C3, and anti-TCC mouse mAbs. Connecting lines are four parameters nonlinear fitting using the equation inhibitor concentration vs. slope. Data are reported as [(OD inhibitor -OD background )/(OD noinhibitor -OD background )]100. Error bars represent the SEM of three independent experiments, and the dashed line the 50% inhibition level. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Inhibition, Incubation, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: Chimeric and rMAP-1 complement inhibition after co-incubation with NHS. Serial dilutions of MAP-1:C4BP 1−5 (M:C), C4BP 1−5 :MAP-1 (C:M), and rMAP-1 were co-incubated with 2% NHS in non-adsorbent titration plates for 30 min prior to addition to mannan-bound rMBL. Detection of deposition of C4 (A) , C3 (B) , and TCC (C) was determined as described before. Connecting lines are four parameters nonlinear fitting using the equation inhibitor concentration vs. slope. Dashed line Data are reported as [(OD inhibitor – OD background )/(OD noinhibitor – OD background )]100. Error bars represent the SEM of three independent experiments, and the dashed line the 50% inhibition level. * P < 0.05; *** P < 0.001.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Inhibition, Incubation, Titration, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: Cofactor activity in factor I-mediated C4b and C3b cleavage. Purified C3b or C4b was incubated with fI and different cofactors for 2 h at 37°C. The reactions were stopped with LDS buffer and subjected to western blotting under reducing conditions using C4d and C3d antibodies. (A) Incubation of C4b with fI in the presence of C4BP, fH or C4BP-containing chimeric proteins resulted in the generation of a band corresponding to the inactive degradation products iC4b and C4d. (B) Incubation of C3b with fI in the presence of C4BP, fH or C4BP-containing chimeric proteins results in the generation of the 68 kDa degradation fragment. M:C, MAP-1:C4BP 1−5 ; C:M, C4BP 1−5 :MAP-1. The blots are representative of three independent experiments.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Activity Assay, Purification, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: Inhibition of complement deposition on tubular proximal kidney epithelial cells. (A) HK-2 cells were incubated with increasing concentrations of rMBL at 4°C and bound MBL was determined using an anti-MBL mAb. (B) Binding of MBL results in the deposition of C4. HK-2 cells were incubated with rMBL followed by 10% MBL defect serum for 1 h at 4°C. Bound C4 was determined using an anti-C4c pAb. (C) rMBL-mediated C4 deposition after MAP-1:C4BP 1−5 (M:C), C4BP 1−5 :MAP-1 (C:M), rMAP-1 or full length C4BP co-incubation with 10% MBL defect serum. Inhibition is reported as the ratio of the MFI of the inhibitor to the MFI of the bank (MFI inh :MFI blank ). Significance was tested for each concentration of the inhibitors compared to the blank. Data are reported as the mean ± SEM of three independent experiments.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Inhibition, Incubation, Binding Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

doi: 10.3389/fimmu.2018.01945

Figure Lengend Snippet: Wielisa total complement screen. Serial dilutions of MAP-1:C4BP 1−5 (M:C), C4BP 1−5 :MAP-1 (C:M), rMAP-1, rC4BP 1−5 , and rMAP-1 and rC4BP 1−5 together (M+C) were incubated with NHS and subsequently the protein/serum mix was applied to pre-coated Wielisa plates. Complement activation was quantified using an anti-TCC mAb. (A) Lectin pathway activation on mannan-coated plates. (B) Classical pathway activation on IgM-coated plates. (C) Alternative activation on LPS-coated plates. Connecting lines are nonlinear fitting using the equation inhibitor concentration vs. slope. Values represent OD readings normalized to the OD of the wells without inhibitor times 100. Significance was tested for best-fit IC50 values of data sets with an adjusted R-squared > 0.8. Error bars represent the SEM of three independent experiments, and the dashed line the 50% inhibition level. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were probed with 0.5 μg/ml anti-MAP-1 20C4 or 0.3 μg/ml anti-human C4BP polyclonal antibody (pAb; Abnova, 722-D01P, Taiwan) overnight, followed by 1 h incubation with 0.4 μg/ml rabbit anti-mouse-HRP pAb (Dako, P0260, Denmark) or a 1:4000 dilution of donkey anti-rabbit-HRP pAb (GE Healthcare, NA934V) for MAP-1 and C4BP, respectively.

Techniques: Incubation, Activation Assay, Concentration Assay, Inhibition