Topological Locationzation of Epitopes on Hemocyanin by Molecular Immunoelectron Microscopy

Abstract

The intramolecular localization of an epitope on an antigen (AG) can be deduced from the architecture of the immunocomplex (IC) string. Indeed, the epitope is located in the surface portion of the AG in contact with the antibody (AB). The electron microscope is well adapted to the study of the IC architectures, and allows a direct topological localization of epitopes. In this work, the hemocyanin (Hc) of the scorpion Androctonus australis, a 1800 kDa protein comprising 24 polypeptide chains belonging to 8 different types, was used as the AG.Sixteen epitopes were located on the Aa6 subunit using a double approach. First, the architecture of the IC strings composed of native 24-meric Hc and monoclonal antibodies (MABs) was studied by electron microscopy and image processing. Second, the relative positions of the epitopes were determined using IC containing the free subunit.To determine the architecture of IC strings containing 24-meric Hc, whole Hemolecules were incubated with purified MABs specific for the Aa6 subunit, then the ICs were purified by gelfiltration on AcA 34, negatively stained with 2% uranylacetate, and observed in the electron microscope (Fig. 1).