Western blots of interactions of C1-inh, autoanti-C1-inh, and C1s. All protein reagents were in PBS and the reaction volumes were all 40 /xl. (A). Western blot from nonreducing SDS-PAGE. The concentration of C1 -inh was 1.4 jxM while the autoantibody was used at the 50% inhibitory concentration (0.4 /xM). The concentrations of C1s were varied. Reaction time of autoantibody with C1-inh was 2 hr and that of the autoantibody-C1-inh mixture with C1s was 3 hr at 37°C. The reactions were stopped by addition of sample buffer and samples were separated by SDS-PAGE (7.5% w/v Polyacrylamide). Proteins were transferred to nitrocellulose by blotting and detected by goat anti-human C1-inh and rabbit anti-goat IgG conjugated with horse radish peroxidase. C1-inh was detected using ECL reagents and the result is shown. The observed bands are: >190 kDa, C1s-C1-inh complex; 115 kDa, intact C1-inh; 96 kDa, a cleaved form of C1-inh. Lane 1: C1-inh alone; lanes 2-11: C1-inh and Patient 1 autoantibody incubated for 2 hr, 37°C, which then continued for a further 3-hr incubation with different amounts of C1s: 0.3 µM (lane 2); 0.4 µM (lane 3); 0.6 µM (lane 4); 0.7 µM (lane 5); 0.8 µM (lane 6); 1.0 µM (lane 7); 1.2 µM (lane 8); 1.3 µM (lane 9); 1.5 µM (lane 10), and 1.6 µM (lane 11). (B) Western blot from nonreducing SDS-PAGE. The concentrations of C1-inh and autoantibody were 2.1 µM and 1.3 µM, respectively, and that of C1s was 8.8 µM in a total reaction volume of 40 µl. The reaction conditions were the same as in A, except that incubation of autoantibody-C1-inh mixture with C1s was for 6 hr at 37°C. Lane 1: C1-inh and C1s; lane 2: C1-inh alone; lane 3: Patient 1 autoantibody and C1-inh and then C1s; lane 4: Patient 2 autoantibody and C1-inh and then C1s. (C) Western blot from reducing SDS-PAGE. Reaction concentrations of C1-inh, autoantibody, and C1s were 2.1 µM, 1.3 µM, and 1.8 µM, respectively. Lane 1: C1-inh alone; lane 2: C1-inh and autoantibody; lanes 3–5: C1-inh and autoantibody were incubated for 2 hr at 37°C, then C1s was added and incubation continued for 1 hr (lane 3), 3 hr (lane 4), and 5 hr (lane 5). This gel does not resolve the 105 kDa and 96 kDa bands effectively, so they appear (lanes 3–5) as a single broad band. Data shown in this figure were obtained with Patient 1 autoantibody but a similar results were obtained with Patient 2 autoantibody. (D) Scanning densitometry of the gel shown in Fig. 4A. As the concentration of C1s increased, the intensity (area under the curve) of the 115 kDa band of native C1-inh decreased, while that of the 96 kDa band (cleaved C1-inh) continued to increase. The intensity of the 190 kDa band (C1s-C1-inh complex) increased to reach a plateau level when the C1s concentration was approximately 1 µM. The density of the 190 kDa band may be an underestimate of the amount of C1s-C1-inh complex formed because of the possibility of inefficient transfer of higher molecular weight species from gel to membrane.