Antibody binding to blood group antigens in relation to temperature: scanning electron microscopy of immunogold‐labelled erythrocytes

Abstract

Binding patterns of mouse monoclonal antibodies (mAb) to P1, Pk, N, I, H, Y or A antigens were visualized in the backscatter electron imaging mode of a scanning electron microscope by indirect immunogold labelling. Experiments were performed at room temperature (RT) and at 4 degrees C. In experiments with anti-P1 and anti-Pk, clusters of immunolabelling particles dominated the immunolabelling pattern much more at RT than at 4 degrees C. By contrast, no clustering was seen with anti-N, even at RT. Clustering was also observed at RT with anti-I, anti-H and anti-Y, and on some Ax and A3 cells with anti-A, but was much reduced at 4 degrees C. Immunolabelling was stronger at 4 degrees C than at RT with all mAb except anti-N and anti-A. The results indicate that glycolipid blood group antigens are more mobile in the membrane of intact erythrocytes at RT than at 4 degrees C, and that the cells bind more antibodies to such antigens at 4 degrees C than at RT. We suggest that antigen immobilization in the cold will reduce cross-linking of antigens and hence increase the number of antibody molecules needed for epitope saturation, leading to increased binding of antibody in the cold. This may be the main reason for cold-enhanced agglutination with human blood group antibodies.

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

“…It is well established by agglutination [l, 231 and immunolabelling [3,4] that there are truly A-negative erythrocytes in individuals of phenotype A3. Immunolabelling studies have shown, furthermore, that cell-to-cell varia-234 HeierMamorWCalkovskA/Sandin/Kornstad A Antigens in Weak A Subgroups tion of antigen amount is a general feature of all carbohydrate blood group antigens [3][4][5]14,24]. Truly A-negative cells may exist even in A2 individuals.…”

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

“…In each experiment, 160-300 cells were counted. Similar semiquantitation of labeling has been carried out previously on single labeled cells Heier et al, 1988Heier et al, , 1992aSoligo et al, 1985).…”

“…All labelings using anti-H were incubated a t 4°C (277"K), since anti-H was found to give clusters of labeling particles when incubated at room temperature, 20°C (293°K). The anti-A was incubated at 20°C (293"K), since no difference in labeling pattern or strength was found between the two temperatures (Heier et al, 1992a). Preparation for SEM (BEI).…”

“…Single labeling patterns have been characterized, in the BE1 mode, to investigate the distribution of ABO related oligosaccharide antigens on erythrocytes. Since human antibodies to these antigens may vary in their fine specificities, monoclonal antibodies reacting with biochemically defined epitopes have been applied in all studies (Heier et al, , 1992aHeier and Namork, 1989). Double labeling of red blood cell antigens has also been published, applying monoclonal antibodies of different classes.…”

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

“…cells carry in addition the 'repetitive A' type 3 chains [2], This is due to molecular differences between the A2 and A1 alleles, which makes the N-acetylgalactosamine transferase in A2 cells less effective than that in Ai cells [18]. Small amounts of type 1 [19] and 4 [20] A chains have been detected on tion of antigen amount is a general feature of all carbo hydrate blood group antigens [3][4][5]14,24], Truly A-negative cells may exist even in A2 individuals. It is therefore not unexpected to find phenotypic variants in which the actual carbohydrate antigen is developed on a few cells only.…”