human complement c5 assaylite  (Assaypro)

Journal: Biomolecules

Article Title: Selective Complement Inhibition in Anti-p200 Pemphigoid: Immune Infiltrate Profiles and Therapeutic Implications Compared to Bullous Pemphigoid

doi: 10.3390/biom16020182

Figure Lengend Snippet: Inhibition of complement fixation by selective inhibitors in an in vitro model of anti-p200 pemphigoid. Human skin cryosections were incubated with heat-inactivated, pooled sera from three anti-p200 pemphigoid patients exhibiting strong C3c/C5-fixating activity, followed by a complement source. Increasing concentrations of selective inhibitors targeting C1s (sutimlimab), C3 (compstatin), C5 (tesidolumab), or C5aR1 (avacopan) were added to the complement source. EDTA and tinzaparin sodium served as positive controls for complement inhibition. BMZ complement fixation (C3c or C5; green) was assessed by IF microscopy and semi-quantified relative to untreated controls (no inhibitor). Sutimlimab and compstatin dose-dependently suppressed C3c deposition, while tesidolumab and avacopan led to almost complete inhibition of C5 complement fixation at all tested concentrations. In contrast, no complement fixation was observed in the presence of normal human serum (NHS). Nuclei were visualized by DAPI (blue) counterstaining. Semi-quantitative analyses were performed using three independent experiments. **** p < 0.0001; ** p < 0.01; * p < 0.05. Scale bar, 100 μm.

Article Snippet: Sections were then stained with FITC-conjugated rabbit anti-human C3c (Dako, Carpinteria, CA, USA) or FITC-conjugated human complement C5 AssayLite ® (AssayPro LLC, St. Charles, MO, USA) antibodies for 1 h at RT.

Techniques: Inhibition, In Vitro, Incubation, Activity Assay, Microscopy

Journal: Biomolecules

Article Title: Selective Complement Inhibition in Anti-p200 Pemphigoid: Immune Infiltrate Profiles and Therapeutic Implications Compared to Bullous Pemphigoid

doi: 10.3390/biom16020182

Figure Lengend Snippet: Targeted inhibition of complement deposition induced by BP autoantibodies in an in vitro model. Human skin cryosections were incubated with pooled, heat-inactivated sera from three BP patients with strong C3c/C5 fixation (green). Selective complement inhibitors, including anti-C1s antibody (sutimlimab), C3 inhibitor (compstatin), anti-C5 antibody (tesidolumab), or C5aR1 inhibitor (avacopan), as well as EDTA and tinzaparin sodium (positive controls for complete complement inhibition) were added alongside hirudin plasma as a complement source. Tissues were stained for C3c or C5 deposition, and percentage inhibition of complement deposition was semi-quantified relative to untreated samples. A dose-dependent inhibition of C3c deposition was observed with compstatin, while all other compounds led to a nearly complete C3c or C5 inhibition along the BMZ at all tested concentrations. Normal human serum (NHS) did not induce complement fixation. Nuclei were visualized by DAPI (blue) counterstaining. Data from three independent experiments were used for semi-quantitative analysis. **** p < 0.0001; * p < 0.05. Scale bar: 100 μm.

Article Snippet: Sections were then stained with FITC-conjugated rabbit anti-human C3c (Dako, Carpinteria, CA, USA) or FITC-conjugated human complement C5 AssayLite ® (AssayPro LLC, St. Charles, MO, USA) antibodies for 1 h at RT.

Techniques: Inhibition, In Vitro, Incubation, Clinical Proteomics, Staining