Human complement factor b

Example 14

CVF-Bb complex is prepared from purified cobra venom factor (1 μM); human Complement factor B and human Complement factor D are available from a commercial source (Complement Technology, Tyler, Tex.). CVF-Bb complex at 3 nM concentration is incubated with test compound at various concentrations for 10 minutes at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human Complement C3 substrate (Complement Technology, Tyler, Tex.) is added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction is stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, is quantified by means of an enzyme-linked-immunosorbent assay (Quidel, San Diego, Calif.) and/or denaturing gel electrophoresis (SDS-PAGE). IC₅₀ values are calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.

Example 14

CVF-Bb complex is prepared from purified cobra venom factor (1 μM); human Complement factor B and human Complement factor D are available from a commercial source (Complement Technology, Tyler, Tex.). CVF-Bb complex at 3 nM concentration is incubated with test compound at various concentrations for 10 minutes at room temperature in PBS pH 7.4 containing 10 mM MgCI2 and 0.05% (w/v) CHAPS. Human Complement C3 substrate (Complement Technology, Tyler, Tex.) is added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction is stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, is quantified by means of an enzyme-linked-immunosorbent assay (Quidel, San Diego, Calif.) and/or denaturing gel electrophoresis (SDS-PAGE). IC₅₀ values are calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.