Monitoring the formation of soluble immune complexes composed of idiotype and anti-idiotype antibodies by electron microscopy.

We have previously used immunoelectron microscopy (IEM) to generate a three-dimensional map of idiotypic (Id) and isotypic epitopes on the Fab arms of HGAC 39 (Roux et al., 1987, Proc. natn. Acad. Sci. U.S.A. 84, 4984-4988), a mouse IgG3 monoclonal antibody (Mab). In this report, we analyse the geometry of the various types of immune complexes formed by the interaction of HGAC 39 with Mab directed against four mapped epitopes. Moreover, by sampling of reaction mixtures over time, we show that the kinetics of each of the subpopulations of immune complexes, as defined by geometric configuration, can be determined. The data show that for each antibody (Ab)-HGAC 39 combination the rate of immune complex formation was greatest during the first 1.5-3.5 min but that additional complexes formed through the remainder of the half hour assay period. As anticipated, complexes composed of even number units predominated (primarily dimers and tetramers) and most of these were in the form of closed rings. The data also suggest that the location and orientation of the epitopes on HGAC 39 to which the monoclonal antibodies were bound has an influence on the types of immune complexes generated. Specifically we observed that those anti-idiotype Abs that bind to the distal tip of Fab arms (i.e. in the CDR) are less likely to produce bivalently associated ringed dimers than antibodies that bind to epitopes that are proximal to the CDR and that project laterally from the surface of the Fab arms. These data are interpreted in terms of restrictions on hinge mediated flexibility and steric inhibition between adjacent Fab arms on HGAC 39.