Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.
A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.
D000818:Animals; D003208:Concanavalin A / Q000032:analysis*; D006041:Goats / Q000276:immunology; D006046:Gold; D006651:Histocytochemistry; D006735:Horseradish Peroxidase; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000032:analysis*; D007074:Immunoglobulin G; D007700:Kinetics; D037102:Lectins / Q000032:analysis*; D008214:Lymphocytes / Q000032:analysis / Q000648:ultrastructure; D008264:Macrophages / Q000032:analysis / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D010059:Ovomucin
D007106:Immune Sera; D007074:Immunoglobulin G; D037102:Lectins; D003208:Concanavalin A; D010059:Ovomucin; D006046:Gold; D006735:Horseradish Peroxidase