Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dominant suppression of inflammation by glycan-hydrolyzed IgG

doi: 10.1073/pnas.1301480110

Figure Lengend Snippet: Disturbance of stable ICs and complement activation. CII (1 mg/mL) and anti-CII mAb CB20 (low affinity, A) or (M2139 (high affinity, B) at 1 mg/mL were mixed together at 1:1 ratio and incubated for 30 min at 37 °C, followed by addition of anti-hapten IgG, either unhydrolyzed (Hy2.15) or EndoS-hydrolyzed IgG (Hy2.15H) at the ratio 1:1:1. Twenty microliters of this mixture were loaded onto the capillary tube in the DLS instrument. Relative sizes of ICs present in the solution are indicated in the x-axis and the y-axis denotes percentage of ICs present in the solution. Each sample was measured five to seven times and the bars represent mean values from two experiments. Error bars indicate ± SEM. (C) Complement activation on CII bound anti-CII antibodies were monitored by measuring C3b deposition. Each bar represents mean values from three experiments ± SD. (D) Deposition of C3b on the cartilage of mice was used as a measure of IC deposition and complement activation after the injection of EndoS-hydrolyzed, unhydrolyzed, or mixed anti-CII IgG. Mouse pups (three or four mice per group) were injected with 1 mg each of unhydrolyzed IgG (M2139 + CIIC2 + UL1), EndoS-hydrolyzed IgG (M2139H + CIIC2H + UL1H), or a mixture of IgGs at 1:1 ratio. In each group, 26–44 joints were scored in total. **P < 0.01; ***P < 0.005. Error bars indicate ± SEM. (E) Paw samples collected 24 h later were stained with biotinylated anti-kappa (Left) or goat anti-mouse anti-C3c antibodies (Right). Joint sections from mice injected with PBS (first row), unhydrolyzed (second row), EndoS-hydrolyzed (third row), or a mixture (1:1) of IgG (fourth row) are shown. Magnification ×20. Arrows indicate C3b deposition within ICs formed on the joint cartilage surface. (F) Diagram illustrating possible binding mechanisms involved in the suppression of arthritis by EndoS-hydrolyzed antibodies.

Article Snippet: Twenty microliters of this mixture were loaded onto the capillary tube in the DLS instrument (Precision Detectors Inc.).

Techniques: Activation Assay, Incubation, Injection, Staining, Binding Assay