human complement component c5a duoset elisa  (R&D Systems)

Journal: medRxiv

Article Title: Differential virulence potential of different clades of multidrug-resistant Klebsiella pneumoniae ST258

doi: 10.64898/2026.03.28.26349612

Figure Lengend Snippet: (A and B) Bacteria recovered after 30-minute exposure of 10 8 CFU/mL to 85% human serum. Limit of detection (LOD) is indicated with a dashed line. (A) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (B) 6 biological replicates per strain were tested. (C and D) C3 binding to bacteria was measured using an indirect ELISA. (C) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (D) 6 biological replicates were tested per strain, each with at least 5 technical replicates. (E) Correlation between C3 binding and serum survival of Clade 2 isolates was assessed using Spearman correlation. (F) Uronic acid was quantified from a selection of Clade 1 (n=6) and Clade 2 (n=10) isolates with 3 biological replicates per isolate. Significance was assessed with Welch’s t-test. (G) Correlation between uronic acid quantity and serum survival of 10 Clade 2 isolates was assessed using Spearman correlation. (H) OD-normalized bacteria were reacted with 5% human serum in PBS for 30 minutes. C5a from the supernatant of the reaction was quantified using a sandwich ELISA. At least two biological replicates per isolate were performed. For panels A-D and H, P ≤ 0.05: *, P ≤ 0.01: **, P ≤ 0.001: *** by Mann-Whitney test.

Article Snippet: C5a quantification was performed using the Human Complement Component C5a DuoSet ELISA from R&D systems.

Techniques: Bacteria, Binding Assay, Indirect ELISA, Selection, Sandwich ELISA, MANN-WHITNEY

Journal: European Journal of Trauma and Emergency Surgery

Article Title: Multifaceted intestinal defense following experimental blunt abdominal trauma

doi: 10.1007/s00068-026-03145-0

Figure Lengend Snippet: Posttraumatic defensive pathway activation and cellular immune response in the murine jejunum. ( A ) Proteome profiler-based pathway analysis revealed activation of immune and defense-related mechanisms. The 15 pathways with the highest degree of regulation are shown. ( B, C ) Histological quantification in jejunal tissue demonstrated a significant increase of CD3 + T lymphocytes in the trauma group relative to controls. ( D ) Gene expression profiling of S100a8 showed a significant post-traumatic upregulation in traumatized mice compared to sham animals. ( E ) In contrast, no statistically significant alteration was observed in interleukin-6 (Il6) expression. ( F ) Plasma levels of C5a were unaltered by traumatic exposure. ( G ) C5a in bronchoalveolar lavage fluid (BALF) was significantly increased in mice 24 h after AT compared to shams. C : n = 180 per group; D-G : n = 4–5 per group. Statistical analysis was performed using the Mann-Whitney U test, with significance defined as p < 0.05. Data are presented as mean ± standard error of the mean (SEM)

Article Snippet: To measure the concentrations of C5a in plasma and BALF, a sandwich ELISA was performed using the C5a ELISA Kit Mouse Duo Set (R&D Systems, Minneapolis, USA).

Techniques: Activation Assay, Gene Expression, Expressing, Clinical Proteomics, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Prognostic potential of C5a as a clinical marker. ( a ) Schematic illustration depicting the informatics analysis conducted on primary glioblastoma (GBM) samples obtained from the Severance cohort. ( b ) Volcano plot showing differentially expressed genes (DEGs) in the C5a High group compared to those in the C5a Low group. Upregulated, downregulated, and insignificantly altered DEGs are indicated. Genes highlighted in white boxes are significantly associated with the tumor microenvironment (TME) and inflammation. ( c ) Heatmap illustrating the expression profiles of statistically significant DEGs (P-adj < 0.05) between the C5a High and C5a Low groups. ( d ) Gene Ontology enrichment analysis for biological processes related to DEGs in the C5a High group versus the C5a Low group. ( e ) Gene set enrichment analysis (GSEA) plot showing the enrichment of hallmark TME and inflammation-related gene sets in the C5a High group relative to the C5a Low group. ( f ) Kaplan–Meier survival curve comparing overall survival between patients with GBM in the C5a High group (n = 61) and those in the C5a Low group (n = 35). Patients in the C5a High group exhibited significantly worse survival outcomes compared to those in the C5a Low group (P = 0.027).

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Marker, Expressing

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Evaluation of cell viability, ATP levels, and molecular changes in glioblastoma tumorspheres treated with C5a-enriched conditioned media and W54011 . ( a , b ) Assessment of cell viability and ATP levels in three glioblastoma (GBM) tumorspheres (TS15-88, TS14-15, and U87) treated with C5a-enriched conditioned media (CM) alone or in combination with W54011 at varying concentrations (0–30 μM). Graphs show the dose–response curve used to calculate IC 50 values (solid bars, control [Cont]; bars with slashed lines, C5a-enriched CM-treated). The “0 μM” W54011 condition refers to treatment with CM in the absence of W54011 (i.e., CM + 0 W54011 ), which served as a positive control to evaluate the full effect of C5a without receptor inhibition. ( c ) Quantification of C5a levels in CM derived from GBM tumorspheres using ELISA. The cells were treated with C5a-enriched CM alone or in combination with W54011 at concentrations ranging from 0–7.5 μM (solid bar, Cont; bar with slashed lines, C5a-enriched CM-treated). ( d ) Western blotting analysis of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at indicated concentrations (0, 2, 5, and 7.5 μM). Blots were probed with antibodies against pro-caspase-3, Bcl-2, Bax, pro-PARP, cleaved PARP, and GAPDH. ( e ) Heatmap illustrating changes in the expression of genes within the GBM amplification marker gene set between GBM tumorspheres treated with C5a-enriched CM alone or with 7.5 μM W54011 . Statistical analysis was performed using one-way analysis of variance, followed by Tukey’s post hoc test. Data are presented as means ± standard deviation, with statistical significance indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) to denote differences between groups or relative to control conditions; NS, not significant.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Control, Positive Control, Inhibition, Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Amplification, Marker, Standard Deviation

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Reduction in stemness and morphological changes in glioblastoma tumorspheres following W54011 treatment in the presence of C5a-enriched conditioned media. ( a ) High-throughput brightfield micrographs of the three glioblastoma (GBM) tumorspheres (TS15-88, TS14-15, and U87) treated with C5a-enriched conditioned media (CM) alone or in combination with W54011 at various concentrations (0–7.5 μM), starting from an initial seeding density of 200 cells. The 0 μM W54011 group represents CM-treated cells without antagonist exposure. ( b ) Frequency of stem-like cells within populations of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at various concentrations (0–7.5 μM), as measured using the extreme limiting dilution assay at 14 days post-seeding. ( c ) Western blotting analysis of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at concentrations of 0, 2, 5, and 7.5 μM. Blots were probed with antibodies against Nestin, Sox2, PDPN, OCT3/4, and GAPDH. ( d ) Heatmap showing changes in gene expression within the GBM stemness marker gene set in GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at 7.5 μM. ( e ) Representative confocal micrographs demonstrating colocalization of GFAP (red) and C5a (green) in GBM tumorspheres treated with C5a-enriched CM alone or in combination with 7.5 μM W54011 . Scale bars = 50 μm. Cont, control.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: High Throughput Screening Assay, Limiting Dilution Assay, Western Blot, Gene Expression, Marker, Control

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Inhibition of invasion by W54011 in glioblastoma tumorspheres exposed to C5a-enriched conditioned media. ( a ) Time-lapse images of spheroid invasion in three glioblastoma (GBM) tumorspheres (TS15-88, TS14-15, and U87) within a matrix gel. Representative images show spheroids at baseline (0 h, left corner) and after 72 h of treatment with C5a-enriched conditioned media (CM) alone or in combination with W54011 at various concentrations (0–7.5 μM). “0 μM W54011 ” indicates treatment with C5a-enriched CM alone. Scale bars = 200 µm. ( b ) Quantification of the invasion area of individual spheroids (solid bar, control [Cont]; bar with slashed lines, C5a-enriched CM-treated). ( c ) Western blotting analysis of the three GBM tumorspheres treated with C5a-enriched CM alone or in combination with W54011 at concentrations of 0, 2, 5, and 7.5 μM. Cell lysates were probed with antibodies against Zeb1, N-cadherin, β-catenin, CD133, CD44, Snail, Twist, and GAPDH. ( d ) Heatmap depicting gene expression changes in the GBM invasion marker gene set in GBM tumorspheres treated with C5a-enriched CM alone or with 7.5 μM W54011 . Statistical analysis was performed using one-way analysis of variance, followed by Tukey’s post hoc test. Results are presented as means ± standard deviation, with significance indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), highlighting differences between groups or compared to control conditions.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Inhibition, Control, Western Blot, Gene Expression, Marker, Standard Deviation

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Effect of W54011 on tumor growth in an in vivo model with C5a-enriched conditioned media. ( a ) Schematic illustrating the xenograft model design and treatment protocol. “Adaptation” denotes a one-week acclimation period following animal arrival. “Bolt” indicates surgical implantation of a cranial guide-screw at week –1, enabling orthotopic cell injection at week 0. Eight-week-old BALB/c nude mice were divided into four groups: TS (n = 8), TS + W54011 (n = 5), TS + CM (n = 8), and TS + CM + W54011 (n = 10). Orthotopic xenografts were established with TS15-88-luciferase cells (5 × 10 5 ) alone or with TS15-88 (2.5 × 10 5 ) and tMSLC0903-01 (2.5 × 10 5 ). Mice were treated with or without W54011 before surgical orthotopic implantation to assess the effects of C5a-enriched conditioned media (CM) and W54011 in vivo. ( b ) Tumor growth was monitored using an IVIS Lumina II in vivo imaging system starting on day 8 and every week thereafter. Bioluminescence images show tumor signals obtained at 13 weeks. ( c ) Representative magnetic resonance imaging (MRI) scans at 18 weeks post-inoculation. MRI was performed once at the experimental endpoint to confirm tumor presence and assess volume. Red lines indicate tumor volumes. ( d ) Bar graph quantifying MRI tumor volumes (open circle, DMSO-treated; open triangle, W54011 -treated; white box, only TS15-88 injected; blue box, TS15-88 and tMSLC09030-1 co-injected). ( e ) Staining with 3,3′-diaminobenzidine was used to assess the expression of C5a, N-cadherin, and vimentin in tumor tissues from the TS, TS + CM, and TS + CM + W54011 groups. The scale bars represent 200 µm (top) and 50 µm (bottom), respectively. ( f ) Kaplan–Meier survival analysis of the mouse models. The P-value measured when comparing TS + CM (n = 5) to TS + W54011 (n = 8) was 0.00053, and that when comparing TS + CM (n = 5) to TS + CM + W54011 (n = 10) was 0.032. Statistical analysis was conducted using one-way analysis of variance, followed by Tukey’s post hoc test. Results are presented as means ± standard deviation, with significance indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001), highlighting differences between groups or compared to control conditions.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: In Vivo, Injection, Luciferase, In Vivo Imaging, Magnetic Resonance Imaging, Staining, Expressing, Standard Deviation, Control

Journal: Scientific Reports

Article Title: Restoring the glioblastoma tumor microenvironment by targeting C5a with the antagonist W54011

doi: 10.1038/s41598-025-30853-1

Figure Lengend Snippet: Schematic illustration of W54011 -mediated restoration of the C5a-altered glioblastoma tumor microenvironment. Tumor mesenchymal stem-like cells (tMSLCs) secrete C5a, which activates C5aR1 signaling in glioblastoma cells, driving enhanced proliferation, stemness, and invasiveness. This C5a-enriched microenvironment promotes tumor progression and malignant phenotypes. Pharmacologic inhibition of C5aR1 with W54011 suppresses C5a-induced downstream signaling and restores tumor cells toward a less aggressive state.

Article Snippet: Following a 24-h incubation period, W54011 was administered at various concentrations in the CM for 72 h. The concentration of C5a was determined using the Human Complement Component C5a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s protocol.

Techniques: Inhibition

Journal: Frontiers in Immunology

Article Title: Reactive oxygen species drive the aberrant immune response to a C. neoformans chitin synthase 3 ( chs3Δ ) mutant

doi: 10.3389/fimmu.2025.1691076

Figure Lengend Snippet: Differential regulation of CXCR2 ligands may drive neutrophil recruitment in the absence of ROS: Male and female C57BL/6 or gp91 phox- mice were inoculated with 10 7 CFUs of HK chs3∆ . BAL was collected from each group at 12 h post-inoculation, as well as a phosphate-buffered saline (PBS) control for each group. (A) LBT4, (B) C5a, (C) CXCL1/KC, and (D) CXCL2/MIP-2α. Data are cumulative from two experiments, with a total of 8 mice per group per time point. Values are means ± standard errors of the means (SEM). (**, P <0.005, ***, P <0.001).

Article Snippet: Cytokines were analyzed using Mouse CXCL1/KC ELISA kit (R&D Systems), Mouse CXCL2/MIP-2α ELISA kit (R&D Systems), and Mouse C5a ELISA kit (R&D Systems).

Techniques: Saline, Control