Labelling of colloidal gold with IgE

Summary Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal ¹²⁵I-IgE antibovine milk -lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1–8.8 (28 nm in particle diameter). At low coverage of the particles (5 molecules per particle), the isotherm was independant of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2–5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mappings of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4°C. Surface clusters developed when the cells were incubated at 37°C.