Interaction of the WT and mutant factor B proteins with factor B natural ligands—C3 and C3b. (A) ELISA (Ai) or SPR (Aii) assay with C3 coated to the plate or to the biosensor chip, respectively. In panel Aii, the kinetic fit for each sensorogram was given in gray. (B) Hemolytic assay (Bi) or SPR analysis (Bii) with C3b-coated erythrocytes or C3b-coated biosensor chip, respectively. Native C3b was generated on the 2 surfaces by C3 convertase. Erythrocytes lysis was induced only in the presence of C3b, factor D, and recombinant WT or mutant BF together. Removing one component from the system abrogated the lysis (data not shown). (C) Hemolytic test for the dissociation of the C3 convertase (C3bBb), formed by the WT or mutant factor B on the surface of the erythrocytes. The results are presented as percentage of the lysis, obtained in absence of plasma (Z∼2), that is, only spontaneous dissociation of the formed C3 convertase. Supernatant from cells transfected with the vector only was used as a control (SN0) for all experiments. For the SPR and the hemolytic tests, 1 representative experiment of 3 to 5 performed is given. For the ELISA average ± SD, n = 3.