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

Abstract

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.

Different Genotypes Causing Indiscernible Patterns of A Expression on A_el Red Blood Cells as Visualized by Scanning Immunogold Electron Microscopy

“…1a). The latter cells were labeled more strongly than any anti-A sensitized, immunogold-labeled cell seen previously in subgroups A l , A 2 , A 3 or A x [17, 19]. However, such very strongly labeled cells have been seen also previously in A m , A el and B weak [19, 20].…”

“…Labeling with anti-H in A 3 was only slightly stronger than in A 2 [19]. The distribution pattern of A in A el (and in A m ) therefore does not seem to be caused by an anomalous development of the A precursor H.…”

Expression of A Antigens on Erythrocytes of Weak Blood Group A Subgroups

“…It is well established by agglutination [1,23] and immunolabelling [3,4] that there are truly A-negative erythro cytes in individuals of phenotype A3. Immunolabelling studies have shown, furthermore, that cell-to-cell varia were agglutinated most strongly by Ax and A2 eluates, weakly by Am eluates and not at all by Ad eluates.…”

“…For the immunolabelling studies the cells were sensi tized with monoclonal blood group antibodies (mAbs) of biochemically characterized specificities, labelled with colloidal gold particles conjugated to anti-antibodies and visualized in the backscatter electron imaging mode of a scanning electron microscope [7][8][9][10]. For comparison and for extension of previous results [3,4], A3 and AN cells were included in the experiments.…”

“…From hyperim mune pregnancy sera Ax, Am and Ad erythrocytes absorbed antibodies which seemed to have other fine specificities than those absorbed by A2 cells. We conclude that weak subgroups of A may deviate from A2 both by number of erythrocytes expressing A antigens and the biochemical nature of the anti gens.weaker subgroups is a matter of antigen density [1], but we have suggested from studies by scanning immunoelectron microscopy of A3 [3,4] and Ax cells [3] that their A anti gens, too, differ biochemically from that on A2 cells. In a similar study of erythrocytes from a Bd or By individual, we found that although 97% of the cells were B negative, about 1% carried large amounts of B antigen [5].…”