Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: A second generation of C3 humanized rats for preclinical evaluation of human C3 inhibitors

doi: 10.1016/j.bvth.2026.100138

Figure Lengend Snippet: C3 inhibitor AMY-101 inhibits the alternative but not classical pathway complement-mediated hemolysis in the second-generation hC3 rats in vitro. (A) The C3 inhibitor AMY-101 (0.3-20μM) was incubated with rabbit RBCs in the presence of 80% WT rat serum, or second-generation hC3 rat serum in Mg 2+ -EGTA buffer at 37°C for 30 minutes, then hemolysis was quantitated, showing that AMY-101 almost completely inhibited the second-generation hC3 rat alternative pathway-mediated hemolysis at a concentration as low as 2.5μM while did not affect the WT rat alternative pathway-mediated hemolysis at a concentration as high as 20μM. (B) The C3 inhibitor AMY-101 (0.3-10μM) were incubated with antibody-sensitized sheep RBCs in the presence of 10% second-generation hC3 rat serum in GVB ++ buffer at 37°C for 30 minutes, then hemolysis was quantitated, showing that AMY-101 did not have any effect on the second-generation hC3 rat classical pathway-mediated hemolysis at a concentration as high as 10μM. 2nd gen, second generation.

Article Snippet: In these assays, human and rat C3 proteins were detected using a complement C3 polyclonal antibody (MP Biomedicals, catalog no. 0855117) and a secondary anti-goat immunoglobulin G (IgG) (heavy chain+light chain [H+L]) polyclonal antibody (SouthernBiotech, catalog no. 6160-05).

Techniques: In Vitro, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Complement inhibition by C3-siRNA treatment prevents AChR loss and reduces complement activation in the rat Passive Transfer Myasthenia Gravis (PTMG)

doi: 10.1101/2025.08.31.673367

Figure Lengend Snippet:

Article Snippet: Next, plates were again washed and 25 μl/well of primary detection goat anti-C3-Biotin antibody ( :1000 in 1% Blocker A buffer, Origene #AP21439BT-N) was added and incubated for 1 h at RT with shaking.

Techniques: Injection, Clinical Proteomics

Journal: bioRxiv

Article Title: Complement inhibition by C3-siRNA treatment prevents AChR loss and reduces complement activation in the rat Passive Transfer Myasthenia Gravis (PTMG)

doi: 10.1101/2025.08.31.673367

Figure Lengend Snippet: A) After baseline measurements on day 0, subcutaneous treatment injections were administered on day 1 and 8. Intermediate testing and blood collection were performed on day 4, 7 and 11 (not shown). On day 15, indicated with a red arrow, PTMG was induced using mAb35, while non-MG animals received a saline injection of the same volume. B-C) Plasma C3 expression was reduced by the sc administration of C3-siRNA at various dose levels in B) non-MG animals and C) PTMG animals. For animals receiving 2 x 30 mg/kg treatment, doses were administered on days 1 and 8. For animals receiving 1 x 10 or 1 x 30 mg/kg doses, administration occurred on day 8, as indicated by the black arrows on the graphs. Saline-treated animals showed no significant change in C3 levels over time. Group averages are shown. D) Plasma C3 level changes in individual treatment groups on day 17, normalized to baseline levels on day 0. Individual plasma C3 levels per animal are shown. Statistical analysis was performed with Two-Way ANOVA (B-D), followed by One-Way ANOVA and post-hoc Bonferroni’s multiple comparison test (D); α=0.05; ****p<0.0001. A) Created in BioRender. Schöttler, A. (2025) https://BioRender.com/b4xxodd .

Article Snippet: Next, plates were again washed and 25 μl/well of primary detection goat anti-C3-Biotin antibody ( :1000 in 1% Blocker A buffer, Origene #AP21439BT-N) was added and incubated for 1 h at RT with shaking.

Techniques: Saline, Injection, Clinical Proteomics, Expressing, Comparison

Journal: bioRxiv

Article Title: Complement inhibition by C3-siRNA treatment prevents AChR loss and reduces complement activation in the rat Passive Transfer Myasthenia Gravis (PTMG)

doi: 10.1101/2025.08.31.673367

Figure Lengend Snippet: C3 protein levels in plasma of healthy animals receiving a single treatment injection to establish the dose range of C3-siRNA for the PTMG study. After baseline measurements on day 0, C3-siRNA was administered on day 1 (black arrow) and plasma C3 protein levels analyzed on days 3, 8, 15, 22, and 29. Per group N=3. Group averages are shown.

Article Snippet: Next, plates were again washed and 25 μl/well of primary detection goat anti-C3-Biotin antibody ( :1000 in 1% Blocker A buffer, Origene #AP21439BT-N) was added and incubated for 1 h at RT with shaking.

Techniques: Clinical Proteomics, Injection

Journal: bioRxiv

Article Title: Complement inhibition by C3-siRNA treatment prevents AChR loss and reduces complement activation in the rat Passive Transfer Myasthenia Gravis (PTMG)

doi: 10.1101/2025.08.31.673367

Figure Lengend Snippet: C3 protein levels in plasma of healthy animals receiving three consecutive treatment injections, with a cumulative dose equal to a single injection as shown in . After baseline measurements on day 0, C3-siRNA was administered on day 1, 2, and 3 (black arrows) and plasma C3 protein levels analyzed on day 3, 8, 15, 22 and 29. Per group N=3. Group averages are shown.

Article Snippet: Next, plates were again washed and 25 μl/well of primary detection goat anti-C3-Biotin antibody ( :1000 in 1% Blocker A buffer, Origene #AP21439BT-N) was added and incubated for 1 h at RT with shaking.

Techniques: Clinical Proteomics, Injection

Journal: bioRxiv

Article Title: Complement inhibition by C3-siRNA treatment prevents AChR loss and reduces complement activation in the rat Passive Transfer Myasthenia Gravis (PTMG)

doi: 10.1101/2025.08.31.673367

Figure Lengend Snippet: Three days after treatment injection, hepatic C3 mRNA expression was significantly repressed in all C3-siRNA treated animals (left). 30 days after C3-siRNA injection, the expression was still reduced to 50% and ca. 25% in animals that received 10 or 30 mg/kg C3-siRNA (single and cumulative) compared to control animals. Each dot indicates one animal. Statistical analysis as performed using One-Way ANOVA with post-hoc Bonferroni’s multiple comparison test; α=0.05; **p=0.003, ***p<0.0004, ****p<0.0001.

Article Snippet: Next, plates were again washed and 25 μl/well of primary detection goat anti-C3-Biotin antibody ( :1000 in 1% Blocker A buffer, Origene #AP21439BT-N) was added and incubated for 1 h at RT with shaking.

Techniques: Injection, Expressing, Control, Comparison

Journal: bioRxiv

Article Title: Complement inhibition by C3-siRNA treatment prevents AChR loss and reduces complement activation in the rat Passive Transfer Myasthenia Gravis (PTMG)

doi: 10.1101/2025.08.31.673367

Figure Lengend Snippet: One PTMG animal treated with 30 mg/kg C3-siRNA showed no siRNA in the liver. Assuming a misinjection of C3-siRNA, this animal was excluded from the analysis. Each dot indicates one animal. Statistical analysis was performed using One-Way ANOVA with post-hoc Bonferroni’s multiple comparison test; α=0.05; ***p=0.0009, ****p<0.0001.

Article Snippet: Next, plates were again washed and 25 μl/well of primary detection goat anti-C3-Biotin antibody ( :1000 in 1% Blocker A buffer, Origene #AP21439BT-N) was added and incubated for 1 h at RT with shaking.

Techniques: Comparison

Journal: bioRxiv

Article Title: Complement inhibition by C3-siRNA treatment prevents AChR loss and reduces complement activation in the rat Passive Transfer Myasthenia Gravis (PTMG)

doi: 10.1101/2025.08.31.673367

Figure Lengend Snippet: A) Grip strength decreased in PTMG animals after disease induction (red arrow) with a dose-dependent effect of the C3-siRNA treatment. B) Grip strength on day 17 with non-MG animals pooled into one group. C) Disease scoring increased in PTMG animals after disease induction (red arrow), with an evident dose dependent effect of the C3-siRNA treatment. D) Disease score on day 17 with non-MG animals pooled into one group. In A) and C) group averages are shown. In B) and D) each dot represents one individual animal; horizontal bars indicate group means. Statistical analysis was performed with Two-Way ANOVA (A-C), followed by One-Way ANOVA and post-hoc Bonferroni’s multiple comparison test (B) or Kruskal-Wallis test (D); α=0.05; *p=0.0408, **p=0.0021, ****p<0.0001.

Article Snippet: Next, plates were again washed and 25 μl/well of primary detection goat anti-C3-Biotin antibody ( :1000 in 1% Blocker A buffer, Origene #AP21439BT-N) was added and incubated for 1 h at RT with shaking.

Techniques: Comparison