Backscatter electron imaging of double immunogold labeled erythrocytes using two primary monoclonal IgM antibodies

The majority of mouse monoclonal antibodies reacting with blood group epitopes on erythrocytes are of the IgM class, have equal light chain type, and are available as culture supernatants only. To study the interrelationship of the blood group antigens, a method is presented which allows double labeling applying two unconjugated monoclonal antibodies of the same class and species. The method comprises two indirect, sequential labelings using mouse IgM anti-A and anti-H as primary antibodies and two goat anti-mouse IgM conjugated to 30 and 20 nm colloidal gold particles as secondary antibodies. After labeling for the first antigen, free binding sites on the primary antibody are blocked by incubation with an unconjugated goat anti-mouse antibody. The free anti-species on the secondary antibody, conjugated to 30 nm gold particles, are inactivated by silver enhancement. The silver enhancement also enlarges the gold particle for optimal discrimination between the two particle sizes, which are chosen accordingly. Semiquantitations of double labeled cells from subgroup A2 and A3 were found to be in good agreement with the counts of the corresponding single labelings as well as between experiments, irrespective of which of the two antibodies was applied in the first labeling sequence. The results were in accordance with a reciprocal but nonlinear relationship between the A and H antigens and suggest different affinities of the two antibodies for the epitopes in the subgroups investigated, indicating different biochemistry of the antigen determinants.