| Assay Method Information | |
| | Human C3bBb Enzyme Assay |
| Description: | Compounds disclosed herein were evaluated for their potency to inhibit human C3bBb in a biochemical assay. To generate active C3bBb complex in vitro, purified human C3b (Complement Technology, A114), CFB (Complement Technology, A135) and CFD (Complement Technology, A136) were mixed in a 1:1:1 ratio to a final concentration of 1 μM each in the C3bBb assay buffer (PBS pH 7.4, 100 μM NiCl2, 0.05% (w/v) CHAPS). The reaction mix was incubated at room temperature for 30 minutes on a rotating platform. After the incubation, a sample was taken to confirm the cleavage of CFB by Western blot using a polyclonal anti-human factor B antibody (Quidel, A311), and the rest of the reaction was immediately aliquoted on ice and stored at −80° C. On the day of the enzyme assay, a reaction mixture containing 3 nM C3bBb complex and 1 μM purified human C3 (Complement Technology, A113) was prepared in the C3bBb assay buffer. Compounds were serial diluted 3-fold in 100% DMSO for a 10-point dose-response curve. A 0.5 μL aliquot of diluted compound solution was transferred to a conical bottom 96-well plate, before 49.5 μL of the reaction mix was added to each well to initiate the reaction. The plate was incubated for 1 hour at room temperature. The reaction was terminated by adding 0.5 μL of Protease Inhibitor Cocktail (Thermo Fisher, 75446) to each well. The generation of C3a from C3 by the C3bBb complex was measured using the MicroVue C3a Plus EIA ELISA kit (Quidel, A031). The concentration of C3a from each well was calculated using a standard curve. Percent inhibition values were generated using the baseline (C3 only) and the maximum control (DMSO instead of compound), and IC50 of each compound was calculated using 4-parameter logistic regression. |
| Affinity data for this assay | |
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