BACKSCATTER ELECTRON IMAGING OF DOUBLE IMMUNOGOLD LABELED ERYTHROCYTES USING 2 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 int errelationship of the blood group antigens, a method is presented whic h allows double labeling applying two unconjugated monoclonal antibodi es of the same class and species. The method comprises two indirect, s equential labelings using mouse IgM anti-A and anti-H as primary antib odies 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 inc ubation with an unconjugated goat anti-mouse antibody. The free anti-s pecies on the secondary antibody, conjugated to 30 nm gold particles, are inactivated by silver enhancement. The silver enhancement also enl arges the gold particle for optimal discrimination between the two par ticle sizes, which are chosen accordingly. Semiquantitations of double labeled cells from subgroup A(2) and A(3) were found to be in good ag reement with the counts of the corresponding single labelings as well as between experiments, irrespective of which of the two antibodies wa s applied in the first labeling sequence. The results were in accordan ce with a reciprocal but nonlinear relationship between the A and H an tigens and suggest different affinities of the two antibodies for the epitopes in the subgroups investigated, indicating different biochemis try of the antigen determinants. (C) 1994 Wiley-Liss, Inc.