Fig. 1

Inhibition of C1s-C1-inh complex formation by autoantibodies. All protein reagents were diluted with PBS-1% Triton ×100 and the reaction volume was set to a total volume of 40 µl. (A) Complete inhibition of C1s-C1-inh complex formation by autoantibody. Fixed concentrations of both C1-inh (2.1 µM) and ¹²⁵I-labeled C1s (1.8 µM) were used in the reactions. Autoantibodies were preincubated with C1-inh for 2 hr at 37°C before addition of C1s. Incubation was continued for a further 2 hr. The reaction was stopped by addition of SDS-PAGE sample buffer and samples were analyzed by SDS-PAGE under nonreducing conditions. An autoradiograph of the dried gel is shown. The observed radioactive bands are: >190 kDa, C1s-C1-inh complex; 85 kDa, C1s; 65 kDa, a form of C1s in which part of the heavy chain is lost (28,29); 27 kDa, light chain of C1s dissociated from intact protein. Lane 1: ¹²⁵I-labeled C1s alone; lane 2: C1-inh and ¹²⁵I-la-beled C1s; lane 3: Patient 1 autoantibody (0.9 µM), C1-inh, and ¹²⁵I-labeled C1s; lane 4: Patient 1 autoantibody (0.9 µM) and ¹²⁵I-labeled C1s; lane 5: Patient 2 autoantibody (0.9 µM), C1-inh, and ¹²⁵I-labeled C1s; lane 6: Patient 2 autoantibody (0.9 µM) and ¹²⁵I-labeled C1s. (B) Inhibition of C1-inh activity by different concentrations of patient 1 autoantibody. The concentrations of C1-inh and ¹²⁵I-labeled C1s used in this experiment were the same as in A. Lane 1: ¹²⁵I-labeled C1s alone; lane 2: C1-inh and ¹²⁵I-labeled C1s; lane 3: autoantibody (0.07 µM), C1-inh, and ¹²⁵I-labeled C1s; lane 4: autoantibody (0.17 µM), C1-inh, and ¹²⁵I-labeled C1s; lane 5: autoantibody (0.33 µM), C1-inh, and ¹²⁵I-labeled C1s; lane 6: autoantibody (0.75 µM), C1-inh, and ¹²⁵I-labeled C1s; lane 7: autoantibody (0.92 µM), C1-inh, and ¹²⁵I-labeled C1s; lane 8; normal IgG (1.0 µM), C1-inh, and ¹²⁵I-labeled C1s.