Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.