Ultrastructural labeling of lymphocyte surface immunoglobulin: The preparation and use of soluble immune complexes as indirect immunoelectromicroscopic markers

Abstract

2 distinct macronuclear markers, ferritin and hemocyanin, may be used in a mixed anti-globulin labeling reaction to localize lymphocyte surface immunoglobulin (Ig) determinants by transmission electron microscopy. Soluble immune complexes of the marker molecules (antigen) are prepared by complexing with specific antiserum in 40 to 50 x antigen excess; uncomplexed Ig is removed by ultrascentrifugation and/or gel filtration chromatography. Immunoelectrophoresis, spectrophotometry and passive hemagglutination inhibition assay are used to determine the purity and amounts of antibody-antigen in the purified immune complexes. For immunoelectron microscopic labeling, the immune complex markers are coupled to lymphocyte surface Ig by an indirect anti-Ig or anti-allotype antibody linkage. Labelled Ig determinants at 0 degrees C or in the presence of sodium azide are visualized as small patches of marker molecules on the lymphocyte surface membrane. This EM labeling method results in much more consistent and generally higher percentages of surface Ig positive cells (60 to 70% of rabbit peripheral blood lymphocytes) than the percentages obtained using other methods, such as immunofluorescence or autoradiography. If the lymphocytes are warmed to 37 degrees C in the absence of azide the labeled surface Ig determinants undergo rapid endocytosis; endocytotic vesicles containing marker molecules are visible. This mixed anti-globulin immunoelectronmicroscopic labeling system may be used to localize a wide variety of antigens on different cell surfaces.