Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture

Journal: PLoS ONE

Article Title: The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

doi: 10.1371/journal.pone.0120411

Figure Lengend Snippet: HMVEC-d were pre-incubated with plasma of patients (N = 30) with acute HSP, plasma of healthy controls (N = 30), or culture medium alone for 48 hr, and then the cells were harvested and analyzed for the expression of C3aR (A) and CD88 (B) by flow cytometry. The expression levels were presented as mean fluorescence intensity (MFI).

Article Snippet: Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: All inductions were performed for 3 days with 1 ×10 6 CD62L hi Foxp3 − CD25 − CD4 + T cells, rhIL-2 (5 ng/ml), and anti-CD3 + CD28 beads (per manufacturer’s instructions), experiments were repeated two times. ( a ) WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 − cells were activated and assayed for percent Foxp3 + CD25 + by flow cytometry (*P<0.001, n=5). (b) Following iT reg induction (as in a ), flow sorted WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were incubated with 10 6 CellTracker Red™ labeled CD25 − Foxp3 − CD4 + WT cells in differing T eff /iT reg ratios + anti-CD3 (5 μg/ml) and 2.5×10 5 CD11c + DCs. Relative suppressive capacity was determined by percent Red-labeled dividers (n=5). (c) WT Foxp3 − CD4 + T cells were induced as in (a) in the absence or presence of anti-C3a (10 μg/ml), anti-C5a (10 μg/ml), or both or in the absence and presence of the antagonists C3aR-A (10 nM), C5aR-A (10 nM), or both. CD4 + T cells then were assayed for Foxp3 expression by flow cytometry (*P<0.001, n=6). (d) Following iT reg induction (as in a ), sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were washed and recultured for 24 hr in the absence of further stimulation. Culture supernatants were assayed for TGF-β, IL-6, and IL-10 by ELISA (*P<0.001, n=6). (e) C3ar1 −/− C5ar1 −/− iT regs were induced (as in a) in the absence and presence of anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Foxp3 + CD25 + T reg percentages were quantified by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Flow Cytometry, Incubation, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: iT reg induction and activation experiments were performed as in with 2.5×10 5 CD11c + DCs and anti-CD3 mAb (5 μg/ml) instead of anti-CD3+CD28 beads, experiments were repeated two times. (a) Sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with WT DCs ± TGF-β1 (5 ng/ml) and assayed for CD25 and Foxp3 expression by flow cytometry (*P<0.001, n=10). (b) (Left) WT Foxp3 − CD4 + T cells were incubated with C3 −/− C5 −/− DCs without TGF-β1 ± C3a/C5a (100 ng/ml) and Foxp3 + CD25 + cells quantified by flow cytometry. (Right) WT Foxp3 − CD4 + T cells were incubated in the presence of TGF-β1 (5 ng/ml) ± C3a/C5a (100 ng/ml) and percent Foxp3 + CD25 + cells quantified (*P<0.001, n=7). (c) Sorted WT or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with C3ar1 −/− C5ar1 −/− DCs in the absence or presence of C5a (100 ng/ml) after which percent Foxp3 + CD25 + cells was quantified. The % Foxp3 + cells with DKO or WT T cells did not significantly differ (P=0.54). (d) Sorted C3 −/− C5 −/− or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with Daf1 −/− DCs ± anti-C3a and anti-C5a mAbs after which percent Foxp3 + CD25 + cells was quantified (*P<0.001; n=6). (e) Sorted WT Foxp3 − or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated for 24 hr either in the absence of DCs or in the presence of WT DCs or C3ar1 −/− C5ar1 −/− DCs. Supernatants then were assayed for TGF-β1 and IL-6 by ELISA (*P<0.001, n=6). (f) C3ar1 −/− C5ar1 −/− Foxp3 − cells were incubated for 3 days with anti-CD3 (5 μg/ml), IL-2 (5 ng/ml), and WT DCs ± anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Following the 3 day induction, Foxp3 + CD25 + T reg percentages were determined by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) WT CD4 + T cells were incubated for 30 min with anti-CD3+CD28 beads after which they were incubated for 10 min with Forskolin (30 μM) ± C3a (100 nM), C5a (100 nM), or both. Levels of cAMP activity were determined by cAMP-Glo assay (*P<0.001). (b) WT, C3ar1 −/− , C5ar1 −/− , and C3ar1 −/− C5ar1 −/− CD4 + T cells were stimulated with anti-CD3 mAb for 30 min after which PKA activity was quantified by PepTag assay. (c) iT regs generated from sorted WT Foxp3 − CD4 + T cells plus TGF-β1, iT regs derived from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells, and conventional Foxp3 − CD4 + T cells were stimulated with anti-CD3+CD28 beads for 15 min and assayed for S 473 and T 308 p-AKT by Luminex assay (n=5). (d and e) WT or C3ar1 −/− C5ar1 −/− CD4 + T cells were incubated for 15 min ± anti-CD3+CD28 beads. Cells were extracted in phospho-lysis buffer and extracts assayed for (d) phospho-rbS6 and (e) phospho-Smad2 by immunoblotting. (f) iT regs induced from WT Foxp3 − CD4 + T cells plus TGF-β1 or from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were assayed for phospho-STAT3 and phospho-STAT5 by flow cytometry (representative plots of n=6).

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Activity Assay, Glo Assay, Generated, Derivative Assay, Luminex, Lysis, Western Blot, Flow Cytometry

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Sorted WT Foxp3 − cells were incubated for 1 hr with anti-CD3+CD28 beads (as in ) in the absence or presence of TGF-β1 (5 ng/ml) and then assayed for complement mRNA transcripts by qPCR (*P<0.001, n=5). (b) Sorted WT Foxp3 − CD4 + T cells were incubated for 1 hr with anti-CD3+CD28 beads plus TGF-β1 (5 ng/ml) alone, TGF-β1 (5 ng/ml) plus IL-6 (5 ng/ml), or IL-6 (5 ng/ml) alone, after which the cells were assayed for C3 mRNA expression by qPCR (*P<0.001, **P<0.02, n=5). (c and d) Sorted WT Foxp3 − CD4 + T cells were incubated for 48 hr with anti-CD3+CD28 beads plus TGF-β1 alone, TGF-β1+IL-6, or IL-6 alone as in (b) . Culture supernatants were assayed for (c) C3a and C5a generation by ELISAs, and (d) C3aR and C5aR surface expression by flow cytometry (P<0.01 for TGF-β1 alone vs TGF-β1+IL-6 or IL-6 alone, Mean Fluorescence Intensity (MFI) values; n=5). (e) Sorted WT Foxp3 − cells were activated for 48 hr with anti-CD3+CD28 beads ± C5a (100 ng/ml) or C3aR-A/C5aR-A (10 nM), after which culture supernatants were assayed for TGF-β1 or IL-6 by ELISA (P<0.05; n=6). (f) Sorted WT Foxp3 − CD4 + T cells were incubated with WT DCs or with C3 −/− C5 −/− DCs in the absence of TGF-β1 ( C3 −/− C5 −/− iT regs ). DCs (left side) were assayed for C5aR and C5L2 expression by gating on CD11c + cells. Responder T cells (right side) were assayed for C5aR and C5L2 expression by gating on Foxp3 − cells in the case of WT CD4 + T cells prepared with WT DCs and on Foxp3 + cells in the case of WT CD4 + prepared with C3 −/− C5 −/− DCs. (*P < 0.01; n=5). (g) Sorted WT or C5L2 −/− (encoded by Gpr77) Foxp3 − CD4 + T cells were incubated with WT DCs (Left) and sorted WT Foxp3 − CD4 + T cells were incubated with WT or C5L2 −/− DCs (right) both in the presence of anti-CD3 and TGF-β1 (5 ng/ml). Percent Foxp3 + CD25 + cells were assayed by flow cytometry. (*P<0.001, n=6). (h) WT Foxp3 − CD4 + T cells were incubated for 3 days with WT DCs plus TGF-β1 (5 ng/ml) after which Foxp3 + and Foxp3 − cells were sorted. Foxp3 − CD4 + T cells or Foxp3 + CD4 + iT regs were incubated for 5 min with anti-CD3+CD28 beads + biotin-labeled C5a, the cells chilled to 4°C, and plasma membrane fractions were isolated by ultracentrifugation. Following C5aR and C5L2 IP, bound proteins were eluted with alkaline solution, and equal amounts of protein (determined by A 280 ) were loaded onto SDS PAGE gels. Following electrophoresis, gels were blotted for biotinylated-C5a by adding streptavidin-HRP followed by ECL reagent.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Labeling, Membrane, Isolation, SDS Page, Electrophoresis

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Flow sorted human CD45RA + CD25 − CD4 + T cells (1×10 6 ) were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous CD11c + DCs in the absence or presence of 1) TGF-β1 (5 ng/ml), 2) each of C3aR-A/C5aR-A (10 nM), or 3) each of anti-C3a/C5a mAbs (10 μg/ml). Percent Foxp3 + CD25 + CD4 + T cells then were determined by flow cytometry. (b) Flow sorted CD25 + cells from (a) were incubated for 3 days with in differing T eff /iT reg ratios with 1×10 6 CFSE labeled autologous naive CD25 − cells, anti-CD3 mAb (3 μg/ml), and 1×10 4 autologous CD11c + DCs. Percent dividers was determined by CFSE dilution. (c) CD45RA + CD25 − CD4 + T cells (1×10 6 ) were isolated from 5 healthy controls (NC) and 3 MS patients by flow sorting. The cells were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous DCs in the absence or presence of 1) rhTGF-β1 (5 ng/ml), 2) C3aR-A/C5aR-A (10 nM), or 3) anti-C3a/C5a mAbs (5 μg/ml). Cells were washed and sorted on CD25. After sorting, CD25 + (T reg ) were incubated for 3 days with anti-CD3 mAb, 2.5×10 5 autologous DCs, and 10 6 CD25 − (Effector) cells prelabeled with CFSE. Percent dividers was determined by CFSE dilution.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Flow Cytometry, Labeling, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

doi: 10.1016/j.jbc.2021.101136

Figure Lengend Snippet: Methods for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , principle of MSD assay to determine C3a desArg formation. B , principle of HTRF assay to detect C3 depletion.

Article Snippet: After evaluation of several complement components and the antibodies against them, the monoclonal antibody 2991 from Hycult Biotech that recognizes the neo-epitope of human C3a/C3a-desArg showed high specificity against C3a desArg.

Techniques: Activation Assay, HTRF Assay

Journal: The Journal of Biological Chemistry

Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

doi: 10.1016/j.jbc.2021.101136

Figure Lengend Snippet: MSD method for determination of complement activation induced by Dezamizumab–SAP complex in human serum. A , specificity of the coating antibody. Purified C3 (■), C3a (●), C3a desArg (○) and iC3b (□) were diluted to various concentrations up to ∼22.2 nM and captured in an MSD plate coated with neo-epitope of human C3a/C3a-desArg monoclonal antibody. In B – D , pooled human serum from Complement Technology was used and the final serum volume was 70% in the complement mixture. B , impact of dilution factor on quantitation of C3a desArg formation yielded from complement activation. Dezamizumab and SAP antigen was mixed at a 4:1 ratio (μg/ml) in the complement activation mixture. The final Dezamizumab concentration was 500 μg/ml (○), 250 μg/ml (●), and serum-only control (□). The reaction was terminated after 40 min at ambient temperature. The complement activation mixture and serum-only control were diluted with HBSP buffer at a range from 5 to 10,935-fold in threefold serial dilution before testing in the MSD assay. C , time course of complement activation at 37 °C. D , time course of complement activation at ambient temperature. In both C and D , Dezamizumab:SAP antigen ratio was 4:1 in μg/ml in the complement mixture. Complement activation was terminated with the stop solution at the indicated time. The complement activation mixtures were diluted 10,000-fold before testing in the MSD method. The MSD signal was back calculated by dilution factors and normalized by subtracting T = 0 signal. The final Dezamizumab concentration was 1000 μg/ml (○), 500 μg/ml (●), 250 μg/ml (□), 125 μg/ml (■), 62.5 μg/ml (Δ), and serum-only control (▲). Error bars for all panels represent the mean ± SD from n = 2 determination.

Article Snippet: After evaluation of several complement components and the antibodies against them, the monoclonal antibody 2991 from Hycult Biotech that recognizes the neo-epitope of human C3a/C3a-desArg showed high specificity against C3a desArg.

Techniques: Activation Assay, Purification, Quantitation Assay, Concentration Assay, Serial Dilution

Journal: The Journal of Biological Chemistry

Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

doi: 10.1016/j.jbc.2021.101136

Figure Lengend Snippet: Effect of Dezamizumab:SAP ratio and concentration on complement activation. MSD method was used. Pooled human serum from Complement Technology was used and the final serum volume was 70%. After 40 min at the room temperature, the complement activation was terminated and diluted 10,000-fold to detect for C3a desArg formation. The Ratio of C3a desArg formation was calculated by sample signal divided by serum-only signal. A , two-fold serial dilutions were performed for Dezamizumab and SAP respectively. The diluted Dezamizumab and SAP were added to the assay plate followed by the addition of human serum with a final volume of 70%. After 40 min at ambient temperature, the complement activation was terminated with the stop buffer. The final Dezamizumab concentration in the complement activation ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM) and SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C3a desArg formation was detected using the MSD method. The signal ratio of complement activation versus an average of serum-only background from eight determinations was plotted against the indicated Dezamizumab concentration at the indicated ratio between Dezamizumab and SAP in molar. Dezamizumab:SAP molar ratios were 84.4 (○), 42.2 (●), 21.1 (□), 10.6 (■), 5.3 (Δ), 2.6 (▲), and 1.3 (▽). B , SAP concentrations without the addition of Dezamizumab. SAP ranged from 0.29 to 150 μg/ml (eq. 0.0012–0.64 μM). C , Dezamizumab concentrations without the addition of SAP. Dezamizumab ranged from 15.6 to 1000 μg/ml (eq. 0.105–6.74 μM). D , Dezamizumab was mixed with SAP at the indicated molar ratio ranging from 3.6 to 9.9. The final Dezamizumab concentration was 500 μg/ml (○) or 250 μg/ml (●). Error bars for all panels represent the mean ± SD from n = 2 determination.

Article Snippet: After evaluation of several complement components and the antibodies against them, the monoclonal antibody 2991 from Hycult Biotech that recognizes the neo-epitope of human C3a/C3a-desArg showed high specificity against C3a desArg.

Techniques: Concentration Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

doi: 10.1016/j.jbc.2021.101136

Figure Lengend Snippet: Minimal human serum component needed for complement activation. Dezamizumab: SAP antigen ratio was 4:1 in μg/ml (molar ratio 6.3:1) in the complement activation mixture. The final Dezamizumab concentration was 100 μg/ml (○) and 250 μg/ml (●). Pooled human serum from Innovative Research was used. The final serum volume in the complement activation mixture ranged from 10% to 70%. After 45 min at ambient temperature, complement activation was terminated and diluted 3645 and 10,935-fold respectively to detect C3a desArg formation in the MSD method. The ratio of C3a desArg formation was calculated by sample signal divided by corresponding serum-only signal. The ratio of C3a desArg formation from the 3645 and 10,935-fold dilution was plotted against percentage of serum volume in complement activation mixture. Error bars represent the mean ± SD from n = 2 determination.

Article Snippet: After evaluation of several complement components and the antibodies against them, the monoclonal antibody 2991 from Hycult Biotech that recognizes the neo-epitope of human C3a/C3a-desArg showed high specificity against C3a desArg.

Techniques: Activation Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

doi: 10.1016/j.jbc.2021.101136

Figure Lengend Snippet: Relative potency determination between Dezamizumab reference standard and the thermal-stressed samples. Dose–response determination was performed with Dezamizumab reference standard (○), and Dezamizumab stored in a formulation buffer at pH 7.0 for 3 months at 2 to 8 °C (●), 25 °C (□), and 40 °C (■), or reference standard stored at −70 °C (○). Human serum in the complement activation mixture was either prepared from the blood of a single donor ( A and B ) or the pooled serum purchased from Complement Technology ( C and D ). C3a desArg formation by MSD method ( A and C ) and C3 depletion by HTRF method ( B and D ) were determined. The twofold serial dilution was performed with a fixed Dezamizumab:SAP molar ratio of 6.3:1. The final Dezamizumab concentration in the complement activation mixture ranged from 0.007 to 3.36 μM. The final serum volume in the complement activation mixture was 40%. The complement activation time was 40 min for A , C , and D , and 10 min for B at the ambient temperature. The complement mixture was diluted 10,000-fold for A and C in the MSD method, and 100-fold for B and 40-fold for D in the HTRF method. The ratio of C3a desArg formation in Dezamizumab samples to serum-only control was plotted against Dezamizumab concentration in the MSD method. Similarly, the %C3 remained in Dezamizumab samples versus the serum-only control was plotted against Dezamizumab concentration in the HTRF method. The average of n = 2 data with ± SD was fit using the full four-parameter EC50 model as described in . Error bars represent the mean ± SD.

Article Snippet: After evaluation of several complement components and the antibodies against them, the monoclonal antibody 2991 from Hycult Biotech that recognizes the neo-epitope of human C3a/C3a-desArg showed high specificity against C3a desArg.

Techniques: Activation Assay, Serial Dilution, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

doi: 10.1016/j.jbc.2021.101136

Figure Lengend Snippet: Schematic overview of the complement cascade illustrating the three activation pathways (classical, lectin, and alternative). The classical pathway is activated when C1q binds to antibody complexed with antigen. The interaction activates C1r and C1s, which cleave C4 and C2. The lectin pathway is activated when mannose-binding lectin (MBL) binds to conserved pathogenic carbohydrate motifs. The interaction activates the MBL-associated serine proteases (MASPs) and cleaves C4 and C2. The products from C4 and C2 cleavage form the classical and lectin pathway C3 convertase, C4bC2a, which cleaves C3 into C3b and C3a. C3b associates with C4bC2a to form the C5 convertase of the classical and lectin pathways, C4bC2aC3b. The alternative pathway (AP) is activated by spontaneous hydrolysis of C3. In the presence of Factors B and D, the AP C3 convertase (C3bBb) and AP C5 convertase (C3bBbC3b) are eventually formed. C5 convertase from all three pathways cleaves C5 to form C5a and C5b. C5b together with C6-C9 forms the membrane attack complex (MAC) that lyses targeted pathogens or damaged self-cells.

Article Snippet: After evaluation of several complement components and the antibodies against them, the monoclonal antibody 2991 from Hycult Biotech that recognizes the neo-epitope of human C3a/C3a-desArg showed high specificity against C3a desArg.

Techniques: Activation Assay, Binding Assay