The binding of complement component C3 to antibody-antigen aggregates after activation of the alternative pathway in human serum

Abstract

Preformed immune aggregates, containing antigen and IgG or F(ab'')2 rabbit antibody, were incubated with normal human serum under conditions allowing activation of only the alternative pathway of complement (C). Both the IgG and F(ab'')2 immune aggregates bound C3b, the activated form of the complement component C3, in a smaller manner. A total of 2-3% of the C3 available in the serum was bound to the aggregates as C3b; the rest remained in the fluid phase as inactive C3b or uncleaved C3. C3b was probably covalently bound to the IgG in the aggregates, since C3b-IgG complexes were demonstrated on sodium dodecyl sulfate/polyacrylamide-gel electrophoresis, after repeated washing with buffers containing high salt or boiling under denaturing conditions. Incubation of the C3b antibody-antigen aggregates in buffers known to destroy ester linkages had little effect on the C3b-IgG complexes, suggesting that C3b and IgG might be linked by an amide bond. Two main types of C3b-IgG complexes were found that had apparent MW of 360,000 and 580,000, corresponding to 1 or 2 C3b molecules, respectively, bound to 1 molecule of antibody. On reduction of C3b-IgG complexes, the .beta.-chain, but not the .alpha.''-chain, of C3b was released along with all the L chain of IgG but only about 1/2 or less of the H chain of IgG. During activation of the alternative pathway of C by immune aggregates containing IgG antibody, the .alpha.-chain of C3b apparently may become covalently bound at 1 or 2 sites in the Fd portion of the H chain of IgG.