Specific Agglutination and Precipitation I. The Mechanism of the Reactions

Field: Immunology and Microbiology (Life Sciences)

Summary and Discussion Agglutinating and precipitating antibodies are a specifically altered fraction of the serum globulin. The antigen-antibody complex, whether sensitized red cells, agglutinated bacteria, or the precipitate formed by a soluble protein and the corresponding antiserum, contains this antibody globulin, demonstrable chemically, immunologically, and by a change in the cataphoretic, flocculating, interfacial, and complement-fixing properties of the antigen towards those of the protein with which it has combined. In the case of the cellular antigen, this antibody is present as an invisible film of specifically adsorbed protein, while in the precipitation reaction, it may constitute the bulk of the material formed. In both cases, the originally hydrophilic globulin has become water-insoluble (denatured), upon combination with antigen. This change in properties is not a phenonomen peculiar to the immune reactions, but is a commonly observed and as yet unexplained property of adsorbed proteins, responsible for their sensitizing effect upon otherwise stable colloidal suspensions. It is suggested that in the case of the immune reactions, this denaturation of the antibody globulin is due to the fact that its specificity is determined by hydrophilic groups. When these combine with antigen, hydrophobic groups necessarily face the water phase, determining the surface properties of the antigen-antibody complex. But when normal serum protein is adsorbed, since there are no groups with a specific affinity to antigen, the molecules naturally orient themselves at the interface so that the hydrophilic groups face the water, and the adsorbed protein acts as a protective film away from its isoelectric point. There are therefore three factors which determine specific flocculation: (1) The hydrophilic antigen is covered, with (2) a film of immune globulin, denatured by its combination with antigen. In the absence of electrolytes the charge due to the ionization of this protein suffices to prevent aggregation. Minute concentrations of (3) electrolyte, however, depress this surface charge below the critical value necessary for stability. The resultant aggregation is therefore primarily of the immune globulin surfaces, and only incidentally of the associated antigen. With insufficient immune-serum, only a very small portion of the cell surface is covered with antibody globulin; most of the impacts are between hydrophilic antigen surfaces, ineffective in producting cohesion. The more immune serum, the greater is the proportion of antigen surface covered with the sensitizing denatured protein, and the correspondingly greater the proportion of effective impacts. The optimum hydrogen ion concentration for flocculation is intermediate between that of the original cell and that of the antibody globulin, shifting towards the latter as the degree of sensitization is increased (i.e., more extensive antibody film). At the optimum reaction, ionization, and therefore the surface charge, are minimal: no added electrolytes are necessary to produce aggregation. In more acid or more basic reaction the surface charge due to the ionization of the adsorbed protein causes a mutual repulsion of the particles; but traces of electrolytes depress this charge and allow the cohesion of the denatured antibody films. The flocculating ion is always the one opposite in charge to the ionized protein, and its flocculating efficiency increases enormously with increasing valence. The further from the isoelectric zone, the greater is the degree of ionization, and the more electrolytes are necessary to depress the surface charge below the critical value. As will be shown in a forthcoming paper, the kinetics of specific flocculation, the so-called “Danysz” and “zone” phenomena, and the varying proportions of antigen and antibody in the aggregates formed, are all in keeping with the theory of specific aggregation as just presented.