Principles of novel complement convertase assay.

Classical complement convertases are assembled on sheep erythrocytes sensitized with antibodies and incubated with C3 or C5-depleted serum. The lack of a particular component allows the complement cascade to proceed only to the stage of classical C3 convertase (C4b2a) or classical C5 convertase (C4b2a3Cb). Low serum concentration and sensitization with antibodies enable the classical complement pathway. Formation of alternative C3 (C3bBb) and C5 (C3bBbC3b) convertases is performed on rabbit erythrocytes upon treatment with moderate concentration of C5-depleted serum in Mg –EGTA buffer (sequestering calcium but not magnesium). Addition of guinea pig serum (as a source of missing complement component as well as MAC components) diluted in EGTA initiates lysis only from preformed convertase complexes since chelating of divalent cations prevents disables de novo convertase formation. Therefore, the amount of released hemoglobin is proportional to the initial number of active convertases formed on erythrocytes.