Studies on the Mechanism of Complement Activation by Murine Immunoglobulin G

UPC-10 myeloma protein, a murine immunoglobulin of the IgG class, and its carbohydrate antigen, levan, have been used to define the nature of classical complement activation by IgG. A four step purification procedure was developed for the myeloma protein to minimize albumin contamination and to insure the structural integrity of the purified antibody. In addition, methods were developed for the isolation of levan from perennial ryegrass and Aerobacter levanicum; a novel technique employing water-soluble sulfonated polystyrene was used to prepare levan oligosaccharides which were then fractionated according to molecular weight. Association constants were determined for the binding of various levan preparations to UPC-10 myeloma protein, thereby allowing an initial understanding of the antigen-antibody interactions observed in this system. Further experiments explored the role of antigen-antibody complexes in classical complement activation. An extremely sensitive assay of hemolytic complement activity employing radiolabeled erythrocytes was developed to allow the use of limited quantities of antigen in these studies. Mixtures of antibody and bacterial levan were found to fix complement only in strictly defined concentration ratios, while no complement fixation was observed when conditions of antigen-excess were present, thereby establishing the aggregation of antibody molecules as a necessary, if not sufficient, step for this activity. A more detailed analysis with sized levan oligosaccharides confirmed this requirement for aggregation and indicated that the molecular weight of the levan oligosaccharides is an important determinant of its ability to assemble antibody molecules into active complexes. Direct observations of such antigen-antibody complexes were made through the use of analytical ultracentrifugation, establishing that all of the levan oligosaccharides capable of inducing complement fixation by IgG molecules were also able to assemble the antibody molecules into multimeric antibody complexes. Thus, these studies find no evidence for a purely conformational mechanism for the expression of complement activation by murine IgG, and instead confirm that aggregation of antibody molecules is required for this effector function.