Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Antibodies for flow cytometric analysis on CiGEnCs.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques:

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Baseline characteristics of aABMR, aTCMR, and NR patients.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Transplantation Assay

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Detailed immunological, histopathological and clinical information of individual patients included.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Tandem Mass Spectroscopy

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Complement Levels in Plasma of aABMR, aTCMR, and NR patients. Plasma levels were measured for C3 (A) , C3d (B) , and C5b-9 (D) . Ratios were calculated from respective C3 and C3d levels (C) , indicating systemic complement consumption. Circles represent individual patients with open circles indicating C4d-positive patients and filled circles symbolizing C4d-negative patients. P values depict results from Mann-Whitney U statistical analysis. Statistical significance is defined as P < 0.05. aABMR, active antibody-mediated Rejection; aTCMR, acute T-cell mediated rejection; NR, non-rejection; ns, not significant.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Semiquantitative scores for C3d and C5b-9 deposition in renal biopsy specimens of aABMR, aTCMR, and NR patients. Semiquantitative scores range from 0 to 4, with higher scores indicating increased positivity in biopsy. Groups are compared regarding glomerular C3d deposition (A) , peritubular capillary C3d (B) , glomerular C5b-9 (C) , and peritubular capillary C5b-9 (D) deposition. Data represent staining results of individual patients. Error bars represent median with interquartile range. Unfilled circles symbolize biopsies of C4d-positive patients, filled circles C4d-negative patients. P-values are derived from Mann-Whitney U tests with statistical significance defined as *P < 0.05, **P < 0.001; aABMR, active antibody-mediated rejection; aTCMR, acute T-cell mediated rejection; NR, non-rejection; ns, not significant.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining, Derivative Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Renal biopsy staining for complement factors in a KTR diagnosed with C4d-positive, aABMR. Selected section of biopsy slide stained for C3d in glomeruli (A) and in peritubular capillaries (B) . Selected section of biopsy slide stained for C5b-9 in glomeruli (C) and in peritubular capillaries (D) . White arrows indicate glomeruli and black arrows indicate peritubular capillaries. Double-compound arrows indicate C5b-9 negative peritubular capillaries (D) . Semi-quantitative scores (0-4) are indicated in the lower right corner of each picture. KTR, kidney transplant recipients; aABMR, active antibody-mediated rejection; s, semi-quantitative score.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: CD59 staining in vivo . Renal biopsy staining for complement regulator CD59 in glomeruli (A, C) and peritubular capillaries (B, D) . Staining was performed on human kidney before transplantation as control (A, B) , on biopsy specimen from a C4d-positive (C4d+) aABMR (C, D) . Black arrows point to CD59-positive peritubular capillaries, double-compound arrows to CD59-negative peritubular capillaries. aABMR, active antibody-mediated rejection.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Staining, In Vivo, Transplantation Assay

Journal: Frontiers in Immunology

Article Title: Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

doi: 10.3389/fimmu.2022.845301

Figure Lengend Snippet: Complement system activation on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis. Complement factors C3 (activated), C4d, and C5b-9 on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis are depicted in (A) with the four different incubation conditions plotted on the y-axis. Deposition of complement regulator, CD59, was measured in flow-cytometry under five different incubation conditions (B) . cABO, ABO-compatible; HLA, Human Leucocyte Antigen; Abs, antibodies; IgG, Immunoglobulin G; FITC, Fluorescein isothiocyanate; iABO, ABO-incompatible.

Article Snippet: Primary antibody , Monoclonal Mouse anti-human activated C3 antibody, which recognizes C3b, iC3b, and C3c fragments (Clone bH6, HM2168S, Hycult biotech, Uden, The Netherlands) 1:50 , Mouse anti-human C4d antibody (Clone 12D11, HM2229 20UG, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human neoantigen-C9 antibody (HM2264, Hycult biotech, Uden, The Netherlands) 1:100 , Mouse anti-human CD59 (HM2120, Hycult biotech, Uden, The Netherlands) 1:100.

Techniques: Activation Assay, In Vitro, Incubation, Flow Cytometry

Journal: Frontiers in medicine

Article Title: Overactivation of the complement system may be involved in intrarenal arteriolar lesions in IgA nephropathy.

doi: 10.3389/fmed.2022.945913

Figure Lengend Snippet: FIGURE 1 Three pathways of complement activation. The classical pathway is activated by IgG– and/or IgM–containing immune complexes. The lectin pathway requires a particular sugar moiety pattern to be recognized and bound by MBL/Ficolins/Collectin 11, leading to a classical pathway–like activation cascade. C4d represents the activation of classical pathway or/and lectin pathway. The alternative pathway is constantly initiated by spontaneous hydrolysis of C3 [C3b (H2O)] that is efficiently powered by the covalent attachment of C3b on an activating surface. Complement factor H (FH) and factor H related protein 5 (FHR5) are regulator proteins of alternative pathway. Three pathways lead to formation of C3 convertase and converge at the C3 level. The addition of C3b to the C3 convertase creates C5 convertase, which triggers the formation of the terminal pathway complete complex (C5b-9), which is also known as membrane attack complex.

Article Snippet: Detail staining protocols were optimized for frozen renal biopsy tissue and formalin-fixed paraffin-embedded renal biopsy tissue with the following antibodies: mouse monoclonal anti-human FH antibody (Santa Cruz, United States), and rabbit polyclonal antihuman FHR-5 antibody (GeneTex, California, United States), mouse monoclonal anti-human MBL antibody (clone 3E7) (Hycult Biotech, United States), rabbit polyclonal anti-human C4d antibody (Bio-Rad, United States), mouse monoclonal anti-human C5b-9 antibody (Abcam, United States), rabbit polyclonal anti-human C3c (Dako, Glostrup, Denmark), rat monoclonal anti-human Gd-IgA1 antibody (KM55) (Immuno-Biological Laboratories, Japan) (11, 15).

Techniques: Activation Assay, Membrane

Journal: Frontiers in medicine

Article Title: Overactivation of the complement system may be involved in intrarenal arteriolar lesions in IgA nephropathy.

doi: 10.3389/fmed.2022.945913

Figure Lengend Snippet: FIGURE 7 Positive staining for MBL (A,G), C4d (B,H), FH (C,I), FHR5 (D,J), C3c (E,K), and MAC (F,L) by immunohistochemistry and immunofluorescence, respectively, along intrarenal arterioles in the arteriolar lesions group of IgAN. (Original magnification ×200).

Article Snippet: Detail staining protocols were optimized for frozen renal biopsy tissue and formalin-fixed paraffin-embedded renal biopsy tissue with the following antibodies: mouse monoclonal anti-human FH antibody (Santa Cruz, United States), and rabbit polyclonal antihuman FHR-5 antibody (GeneTex, California, United States), mouse monoclonal anti-human MBL antibody (clone 3E7) (Hycult Biotech, United States), rabbit polyclonal anti-human C4d antibody (Bio-Rad, United States), mouse monoclonal anti-human C5b-9 antibody (Abcam, United States), rabbit polyclonal anti-human C3c (Dako, Glostrup, Denmark), rat monoclonal anti-human Gd-IgA1 antibody (KM55) (Immuno-Biological Laboratories, Japan) (11, 15).

Techniques: Staining, Immunohistochemistry

Journal: Transplantation Direct

Article Title: Complement Markers in Blood and Urine: No Diagnostic Value in Late Silent Antibody-Mediated Rejection

doi: 10.1097/TXD.0000000000000915

Figure Lengend Snippet: Prediction of biopsy results by DSA characteristics and complement markers. ROC curve analysis of DSA characteristics (A,B), CH50 and complement markers in peripheral blood (C,D), or creatinine-adjusted complement products in urine (E,F) was performed in comparison with the finding of biopsy-confirmed AMR (A,C,E) or C4d staining in PTC, respectively (B,D,F). Panels show ROC curves and corresponding AUC values for each comparison. AMR, antibody-mediated rejection; AUC, area under the curve; CH50, 50% hemolytic complement activity; DSA, donor-specific antibody; MFI, mean fluorescence intensity; PTC, peritubular capillaries; ROC, receiver operating characteristic.

Article Snippet: Beads were then washed and incubated with biotin-conjugated monoclonal antibodies against human C4d or C3d (Quidel, San Diego, CA), and in a second step, phycoerythrin-conjugated Streptavidin.

Techniques: Staining, Activity Assay, Fluorescence

Journal: Transplantation Direct

Article Title: Complement Markers in Blood and Urine: No Diagnostic Value in Late Silent Antibody-Mediated Rejection

doi: 10.1097/TXD.0000000000000915

Figure Lengend Snippet: Death-censored graft survival according to biopsy results (A), urinary protein/creatinine (P/C) ratio (B), C4d in blood (C), and C4d detection in urine (D). The Mantel-Cox log-rank test was used to compare survival rates between groups. For 1 recipient (no biopsy-confirmed AMR), no adequate material for urinary analysis of complement proteins was available. AMR, antibody-mediated rejection.

Article Snippet: Beads were then washed and incubated with biotin-conjugated monoclonal antibodies against human C4d or C3d (Quidel, San Diego, CA), and in a second step, phycoerythrin-conjugated Streptavidin.

Techniques:

Journal: Scientific Reports

Article Title: Classical and alternative complement activation on photoreceptor outer segments drives monocyte-dependent retinal atrophy

doi: 10.1038/s41598-018-25557-8

Figure Lengend Snippet: Photoreceptor outer segments activate both the classical and alternative complement pathway. ( a ) Flow cytometry of C3, C4 and C9 neoepitope proteins on the surface of bovine POS following incubation with normal human serum (NHS, shaded area in histograms) vs C3, C4, C9 depleted serum (solid lines in histograms). ( b ) Diagram of molecular components of the AP and CP/MBL complement pathways. C3 and C5 convertases are indicated in blue and red boxes, respectively. ( c , d ) Bovine POS were incubated with human sera depleted of various complement proteins and quantified by flow cytometry using a C3-specific antibody ( c ) or a C9 neo-epitope-specific antibody ( d ), n = 3. ( e and f ) Bovine POS were incubated with mouse wt, C3 deficient, C4 deficient, or Ig deficient ( Rag2 −/− ) serum, surface stained for C3 ( e ) or C4 ( f ) protein and quantified by flow cytometry. ( g ) Flow cytometry analysis of cell-surface complement regulatory proteins and annexin V binding to bovine POS, solid line is isotype control and shaded area is indicated antibody. ¶ p < 0.0001 NHS vs all other sera, ****p < 0.0001 vs C1q/CFD depleted serum, # p < 0.001 vs mouse wt serum by 1-way ANOVA with Dunnet’s multiple comparisons test. Error bars are SD for triplicate repeats, the experiments were repeated three times with similar results. NHS = normal human serum, CFD = complement factor D, CFB = complement factor B, P = properdin, MFI = mean fluorescence intensity.

Article Snippet: POS were washed 2x in FACS buffer (PBS/0.5% BSA/2 mM EDTA) and stained with either 1 μg/ml mouse-anti-C9 neo-epitope (Hycult clone aE11), mouse-anti-C4d (Quidel), or 1:400 FITC-conjugated goat-anti-C3 (MP Biomedicals) followed by anti-mouse-A647 (Jackson Immunoresearch).

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

Journal: Frontiers in Immunology

Article Title: The Hidden Side of Complement Regulator C4BP: Dissection and Evaluation of Its Immunomodulatory Activity

doi: 10.3389/fimmu.2022.883743

Figure Lengend Snippet: PRP6-HO7 is devoid of complement inhibitory activity. Schematic drawing of C4BP(β+) and C4BP(β-) cofactor activity for factor I-mediated splitting of C4b. Factor I cleaves the α′-chain of C4b at two sites. Partial cleavage generates fragments α3-C4d (70 kDa) and α4 (14 kDa). Further cleavage of α3-C4d yields the small C4d (45 kDa) fragment which remains associated with targets. C4BP(β+), C4BP(β-) and PRP6-HO7, at the indicated concentrations (lanes 3-7), were incubated with C4b (8.9 μg/ml) followed by addition of factor I (4.4 μg/ml). Reaction controls included C4b alone, and C4b + FI. All reactions were stopped after 30 min with SDS-reducing sample buffer. C4b cleavage fragments were separated by 4-12% SDS-PAGE under reducing conditions followed by Western blotting using an anti-C4d MoAb. Red arrows indicate the size of the C4b fragments. Cofactor activity was confirmed by the appearance of α3-C4d (70 kDa) or C4d (45 kDa). Results are representative of 3 independent experiments.

Article Snippet: Complement C4b (8.9 μg/ml) and factor I (4.4 μg/ml) (Merck, Darmstadt, Germany) were mixed with C4BP isoforms (0.6 nM or 6 nM) in low-salt buffer (25 mM phosphate buffer pH 7.4 and 25 mM NaCl) in a total volume of 60 μl and incubated at 37°C for 30 min. Then, reducing SDS sample buffer was added, C4b fragments were separated on a 4–12% gradient SDS-PAGE (NuPAGE Bis-Tris 4-12% Mini Gels, ThermoFisher), and Western blot analysis of C4b fragments was performed using a 1:2000 dilution of Anti-Human C4d MoAb (Quidel, San Diego, CA) overnight at 4 °C, followed by a 1:2000 dilution of polyclonal Goat Anti-Mouse IgG HRP (P0447, Dako) in TBS-Tween 0.05%, 1% BSA, 0.02% NaN 3 .

Techniques: Activity Assay, Incubation, SDS Page, Western Blot

Journal: Frontiers in Immunology

Article Title: Thrombotic microangiopathy mediates poor prognosis among lupus nephritis via complement lectin and alternative pathway activation

doi: 10.3389/fimmu.2022.1081942

Figure Lengend Snippet: Frozen sections for C4d using immunofluorescence among MCD (A, D) , LN (B, E) , and TMA (C, F) patients. (A, D) Staining for C4d was negative in MCD renal tissues. (B, E) Moderate granular positive staining for C4d along the glomerular capillary and arteriole in LN renal tissues. (C, F) Strong granular positive staining for C4d along the glomerular capillary and arteriole in TMA renal tissues. Scale bars represent 50μm.

Article Snippet: Frozen kidney tissue sections were incubated with rabbit anti-human MBL polyclonal antibodies (1:20 dilution, Abcam, Cambridge, UK), rabbit anti-human MASP1/3 polyclonal antibodies (1:20 dilution; Proteintech, Wuhan, China), rabbit anti-human C4d polyclonal antibodies (1:300 dilution; Quidel, America), rabbit anti-human CFB polyclonal antibodies (1:20 dilution, Abcam, Cambridge, UK), and rabbit anti-human CFD polyclonal antibodies (1:20 dilution; Proteintech, Wuhan, China) for 2 hours at 37°C in a moist chamber.

Techniques: Immunofluorescence, Staining