EA rosettes using IgG monoclonal antibody-coated erythrocytes: degree of rosette formation correlates with the amount of antibody on the erythrocytes.

The EA (Fc receptor) rosetting assay, which utilizes sheep erythrocytes (SRBC) coated with anti-SRBC antibodies as indicator cells (EA) is a rapid and convenient method for the detection of Fc-receptor-positive cells. To prevent haemagglutination of the EA indicator cells, it has been necessary to dilute conventional SRBC antisera. Owing to this technical restriction, it has not been possible to determine accurately the contribution of antibody density on the EA indicator cells to the level of Fc-receptor-positive cells measured in the EA rosetting assay. However, the availability of high-titred non-haemagglutinating monoclonal anti-SRBC antibodies has provided a means of examining this problem. Four non-haemagglutinating monoclonal anti-SRBC IgG preparations-two of the IgG2a subclass, an IgG2b subclass-specific and an IgG1 subclass-specific antiserum-were used to coat SRBC at antisera dilutions ranging from 1/20 to 1/2000. The amount of antibody bound to the SRBC was determined by an indirect radioimmunoassay utilizing 125I-labelled protein A. The four monoclonal anti-SRBC antibodies were shown to have unique affinities for the erythrocytes, and each was specific for antigens present in differing amounts on the SRBC. The number of Fc-receptor-positive cells detected in a spleen cell suspension or in a homogeneous Fc-receptor-positive tumour cell population by the EA (Fc) rosetting assay was found to be directly proportional to the amount of monoclonal antibody (regardless of the IgG subclass) bound to the EA indicator cells.