Inhibition of CAP and CCP activity by TT30, fH and an anti–human C5 monoclonal antibody. (A) Inhibition of CAP and CCP after spiking TT30 into human whole blood from 9 donors and measuring activity in processed serum using Wieslab Complement System Alternative ELISA kit (Euro-Diagnostica). Results are expressed as percent baseline level of activity and represent mean ± SEM of duplicates (n = 9). IC₅₀ values for inhibition of CAP and CCP were 0.08 ± 0.03μM and 9.8 ± 7.6μM, respectively, while IC₉₀ values for inhibition of CAP and CCP were 0.14μM and > 29.4μM, respectively. (B) Potency and specificity of targeted CAP inhibition by TT30 compared with factor H. Potency of TT30 and fH spiked into human serum was measured as inhibition of CAP using Wieslab Complement System Alternative ELISA kit. TT30 showed high potency (IC₅₀ = 0.04 ± 0.005 μM) while fH was less effective (IC₅₀ = 3.0 ± 1.6μM). Potency of TT30 and anti–human C5 monoclonal antibody from Quidel Corp. was measured as inhibition of CAP (C) or CCP (D) using Wieslab Complement System Alternative or Classic Pathway ELISA kit. TT30 exhibits selectivity for CAP (IC₅₀ = 0.03 ± 0.007μM in CAP compared with 2.1 ± 0.7μM in CCP) while anti-C5 antibody showed similar activity in either pathway (IC₅₀ = 0.16 ± 0.07μM in CAP compared with IC₅₀ = 0.05 ± 0.009μM in CCP). (E) TT30 was spiked into human serum at 0.3μM alone or in the presence of serial 3-fold dilutions of anti-CR2 monoclonal antibody (clone 1048, starting concentration 6.66μM). Isotype control anti-KLH antibody (BioLegend) was used at the same dilution range to confirm specificity of targeted TT30 activity. TT30-mediated inhibition of MAC formation was unaffected by nonspecific isotype control antibody, while anti-CR2 antibody fully abrogated TT30 activity. Results represent mean ± SEM of duplicate values from 3 independent experiments.