Quantitation of cell membrane antigens at the single cell level. II. On the mechanisms of the dynamic interaction between cell surface and bound ligands.

Abstract

The interaction between cell surface antigens of animals cells and antibodies bound to these were studied by microfluorometry of single cells. This method permitted quantitation of the intensity of immunofluorescence per whole cell and per unit cell surface area. The rate of redistribution and the rate of release of membrane‐bound antibody varied in relation to cell type and kind of antigenic specificity examined. Conditions previously demonstrated to be optimal for capping were used in all test systems. Redistribution was most rapid for immunoglobulin determinants on lymphocytes. Redistribution consisted of a passive patching phase followed by an active capping phase with regard to cell metabolism. The dis‐appearance of membrane‐bound antibody showed a rapid early phase followed by a slow phase in virtually all test systems. The results indicated that redistribution and endocytosis occurred when the membrane‐bound antibodies formed a lattice thus forming multivalent bonds with the cell surface. Univalent binding. of antibody to the cell surface seemed to favour release of the antibody. A suggestive correlation between the rate of redistribution and the rate of release of the same cell surface antigen‐ligand complex was observed. The frequency distribution of the intensity determinations per unit cell surface area was shown to be an objective measure of the pattern of immunofluorescence on the cell membrane. This should permit evaluation of the effect of metabolic inhibitors on the distribution of membrane antigens, The results further indicated that the prozone effect of the intensity curves obtained with indirect immunofluorescence methods occurred only on a mobile cell surface and was caused by unaccessibility of membrane‐bound antibody to conjugate molecules because of cross‐linking and lattice formation or some effect mediated thereby.