human c5a elisa kit  (Elabscience Biotechnology)

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: Advanced Healthcare Materials

Article Title: The Distribution of Complement Proteins in Soft and Hard Coronas Impacts Macrophage Uptake of Nanoparticles

doi: 10.1002/adhm.202503534

Figure Lengend Snippet: Analysis of C3 fragments in FC and HC of PS‐COOH. a) Schematic workflow for analyzing complement activation and C3 deposition of PS‐COOH. Concentration of C5a (b) and C3a (c) in pooled human serum after incubation of PS‐COOH at various surface area concentrations (0.00572–0.572 m 2 mL −1 ). Water served as the negative control, while Zymo (1 mg mL −1 ) was the positive control. d) Schematic representation of C3 activation and processing. Native C3 consists of a β chain (≈72 kDa) and an α chain (≈115 kDa). Upon activation, C3 is cleaved into C3b, in which the α chain appears at ≈110 kDa. C3b is further processed by factor I and factor H, into iC3b. In iC3b, the α chain is composed of two characteristic fragments: α 1’ (≈68 kDa) and α 2’ (≈43 kDa). e) Detection of C3 fragments deposition on PS‐COOH surface using dot blot. f) Western blot analysis of C3 fragments in FC and HC. Bands corresponding to C3 α chain (≈115 kDa), iC3b α 2’ chain (≈43 kDa), and β chain (≈72 kDa). Serum 1 represented serum after PBS treatment. Serum 2 represented serum after Zymo treatment, which induces strong complement activation. g) Quantification of band of iC3b α 2’ chain (43 kDa) using image J. Data are presented as the mean ± standard deviation (n = 3). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human C3a ELISA kits (EH2734) and human C5a ELISA kits (EH0096) were obtained from Wuhan Fine Biotechnology Co., Ltd, China.

Techniques: Activation Assay, Concentration Assay, Incubation, Negative Control, Positive Control, Dot Blot, Western Blot, Standard Deviation

Journal: Advanced Healthcare Materials

Article Title: The Distribution of Complement Proteins in Soft and Hard Coronas Impacts Macrophage Uptake of Nanoparticles

doi: 10.1002/adhm.202503534

Figure Lengend Snippet: Time‐dependent adsorption of C3 fragments in the FC and HC of PS‐COOH. a) Scheme illustrating the preparation of FC and HC. Concentrations of C3a (b) and C5a (c) in human serum after incubation of PS‐COOH for 15, 30, 45, and 60 min. The surface area concentration of NPs in the incubation mixtures was 0.143 m 2 mL −1 . d) Western blot analysis of C3 fragments in FC and HC at different incubation times (1, 5, 10, 15, 30, and 60 min). e) Grayscale quantification of iC3b α 2’ chain (≈43 kDa). Data are presented as mean ± standard deviation (n = 3).

Article Snippet: Human C3a ELISA kits (EH2734) and human C5a ELISA kits (EH0096) were obtained from Wuhan Fine Biotechnology Co., Ltd, China.

Techniques: Adsorption, Incubation, Concentration Assay, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A

doi: 10.1101/2025.01.20.633845

Figure Lengend Snippet: ( A) Restoration of S . Paratyphi A long and O9-antigen increases human C3a and (B) C5a production following incubation with human serum for 60 min at 37°C. The S. Typhi Vi capsule prevents C3a production but does not impact C5a release. Long O-antigen in S. Typhimurium is the primary determinant of C5a release. C3a and C5a release were measured by enzyme-linked immunosorbent assay (ELISA). For statistical comparison, individual replicates are normalized to the corresponding wild-type serovar and statistical analysis performed by one sample t test with a hypothetical value of 1.0, with P values in red indicating statistical significance as P < 0.05. Column bars represent the means, with error bars showing standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A.

Article Snippet: Human C3a and C5a production were measured using the Human Complement C3a ELISA Kit (Invitrogen, catalog #BMS2089) and the Human C5a ELISA Kit (Invitrogen, catalog #BMS2088), respectively, following the manufacturer’s instructions.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Comparison, Standard Deviation

Journal: bioRxiv

Article Title: Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A

doi: 10.1101/2025.01.20.633845

Figure Lengend Snippet: ( A) O-antigen modifications by glycosyltransferases in S. Paratyphi A decrease complement activation as measured by C5a release and (B) IgM binding. (C) and (D) O-antigen modifications by glycosyltransferases in S. Paratyphi A do not impact complement deposition or (E) serum resistance. (F) O-antigen modifications by glycosyltransferases in S. Typhimurium do not impact complement activation, (H) and (I) C3b deposition, or (J) serum resistance, but (G) reduce IgM binding. Statistical analysis was performed by one-way ANOVA on 3-4 independent replicates, with P values in red indicating statistical significance with P <0.05. Column bars represent the means, with error bars displaying standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A.

Article Snippet: Human C3a and C5a production were measured using the Human Complement C3a ELISA Kit (Invitrogen, catalog #BMS2089) and the Human C5a ELISA Kit (Invitrogen, catalog #BMS2088), respectively, following the manufacturer’s instructions.

Techniques: Activation Assay, Binding Assay, Standard Deviation