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Quantitative Measurement of C9 Sites and Their Association to the Ring-Like "Lesions" on Complement-Lysed Membranes: A Morphometric Immunoferritin Study¹

From the Institut für Immunologie und Serologie, Universität Heidelberg, West Germany

Abstract

The appearance of ring-like "lesions" on negatively stained target membranes that had been subjected to complement (C) lysis was observed in the electron microscope more than a decade ago. However, it remained unclear whether the ring-like structures represent micelles or bubbles in the outer leaflet of the membrane or the lytic C5-9 complex itself. In the present work by immunoelectronmicroscopy and morphometric studies the ring-like structures were identified to contain C9, and thus seen to be identical to the proposed C5-9 complex.

EAC 1–9 membranes were incubated with rabbit anti-C9 antibodies followed by ferritin-labeled goat anti-rabbit IgG antibodies. More than 50% of the ring-like structures could be labeled by the staining system. The association of C9 to the C rings was statistically significant (p < 0.001) as measured by two independent morphometric methods. About 50% of the C9-representing label could be observed with a greater distance than 300 Å apart from the C-induced ring-like structures. This finding may suggest that some of the ring-like structures are hidden on the membrane by their unfavorable position or that the ferritin label visualizes C9 not associated with ring-like structures. A possible diversity of the C5-9 complexes is discussed.

Footnotes

¹ This work was supported by Deutsche Forschungsgemeinschaft R 278/1. A short survey had been presented on the Sixth International Complement Workshop, Sarasota, Fla., Nov. 26, 1975, and was published as an abstract in J. Immunol. 116:1747 (1976).