Three-dimensional immunoelectron microscopy of scorpion hemocyanin labeled with a monoclonal Fab fragment

An immunocomplex of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.