Monoclonal antibodies (MoAbs) specific for erythroid antigens will be produced. Three hemagglutinating MoAbs have already been produced by hybridization using intact human red blood cells as the immunogen. Additional immunogens will be employed, including proteolytic fragments of red cell membrane, purified membrane components including glycophorin and band 3, and defined carbohydrate antigens. MoAbs will be selected a) to the same red blood cell membrane antigenic determinant, but having different immunoglobulin class and subclass; b) with different affinities in the same determinant; c) to separate epitopes on the same molecule; d) to either carbohydrate or protein antigens; e) to high and low density antigens on the red cell membrane. These MoAbs will be applied to the study of hemagglutination and the prozone phenomenon. Current understanding of the mechanism of hemagglutination has evolved from experimental work using polyclonal antibodies. We propose to use MoAbs in a quantitative system to evaluate a number of factors, including variable affinity of antibody, antigen site density, antigen mobility, complement, effect of enzymes, ionic strength, and diluents, role of cytophilic IgG, glycoprotein, metabolic inhibitors, temperature, and other variables. The advantage of this model system over previous investigations of hemagglutination and the prozone phenomenon lies in the unique specificity and identical affinity of MoAbs. These properties of MoAbs will be used to answer questions which previously could not be addressed.
