Immune Cytolysis: Electron Microscopic Localization of Cellular Antigens with Ferritin-antibody Conjugates

Immune gamma globulin has been coupled to ferritin by the diisocyanate method of Singer. The final product ("ferroglobulin") had approximately 13 per cent of its gamma globulin coupled to ferritin, and roughly half of its ferritin coupled to gamma globulin. The uncoupled gamma globulin could be removed by ultracentrifugal sedimentation of the free ferritin and the ferritin-antibody conjugates. The characteristics of the native antibody were retained by the ferritin-antibody conjugates, for they could be precipitated by anti-gamma globulin antisera, and when used as antibodies, they reacted specifically with soluble and cellular antigens. Ferroglobulin preparations made from rabbit antisera against whole ascites tumor cells were incubated with the cells, and the location of ferritin determined by electron microscopy of thin-sectioned material. It was found that the immune ferroglobulins localized specifically on antigens of the cell membrane. Some of the ferritin label entered the cells by pinocytosis, but the ferritin-antibody units did not appear able to pass directly through the cell membrane into the cytoplasmic matrix. When cells were incubated with ferritin-labeled antibody and complement, antibody could be located in the cytoplasmic matrix, and it therefore appeared that complement action was required before antibody could pass directly through the cell membrane. This finding was consistent with previous observations that the plasma membrane of an antibody-complement treated cell becomes permeable to large molecules. In broken cell preparations incubated with ferroglobulin, antibody combined with amorphous material and with structures derived from cell membranes and from smooth membranes of the endoplasmic reticulum. The data favor the concept that antigens contained within the membranous structures are most important in the formation of cytotoxic antibodies. The reported experiments support the view that cytotoxic antibodies fix primarily to surface antigens of the cell membrane. The subsequent action of complement establishes the permeability defect that induces the osmotic lysis of the cell and permits antibody to pass into the cell where it may act in a similar fashion on intracellular organelles.