9304790 Roux Immune complexes will be generated by reaction of monoclonal antibodies representing the various human and mouse Ig classes and subclasses with specific antibodies directed against topologically defined determinants. Extensive use will be made of previously investigated systems in which mapped idiotypic and isotypic determinants will serve as probe sites. molecules with altered or mutant hinge regions will be similarly complexed for comparison. Electron micrographic images will be scored for angular relationships and rotational orientations between the various Fab and Fc segments of target and probe molecules. These data will be interpreted in the context of the location of the reactive sites and the class and subclass (i.e., the degree and modes of hinge flexibility) of the reactants. When reaction mixtures are sampled at defined time intervals, a kinetic profile of the formation of immune complexes can also be documented. The goals are: 1) To conduct a comprehensive comparative structural analyses of (a) uncomplexed, and (b) tethered Ig classes and subclasses by EM with emphasis on hinge-imparted flexibility of Fab and Fc arms. 2) To determine the correlation of idiotopes or isotypic determinants to (a) the geometry and (b) the kinetics of immune complex formation as a result of interaction with ligands. 3) To determine the role of specific hinge structures (or other flexibility-imparting segments) of native Ig in the generation of defined immune complex configurations. 4) To determine the effect of genetically engineered alterations of the hinge regions on the formation and geometry of immune complexes. %%% A detailed understanding of the structural and functional aspects of antibodies is of fundamental importance to a wide range of immunological phenomena. One key element that is rarely defined in an antigen-antibody interaction is the relationship between the geometry of the immune complex and its functional attributes. This pro posal describes a series of investigations aimed at studying the three-dimensional geometry of immune complexes as a function of both segmental flexibility and the location of reactive sites (i.e., idiotopes and isotypic determinants). Additional knowledge on the limitations of Ig flexibility and geometry should enhance the conceptual framework in which many basic observations are interpreted. Such information may also have practical applications such as aiding in the development of rational approaches to the design of Ig-based pharmaceuticals. ***
