The specific blocking of an IgG dependent erythrophagocytosis assay by protein G and ELISA determination of in situ bound IgG on erythrocytes of normal donors.

We report the development of an in vitro erythrophagocytosis assay in which the level of phagocytosis reflects the number of IgG molecules bound to the erythrocyte. This assay is sensitive to 300 IgG per erythrocyte above background levels. Blocking the Fc of the bound immunoglobulin with protein G totally blocks macrophage recognition of the opsonized red cell and prevents Fc-gamma-dependent phagocytosis. An accurate, reliable, easily performed CELL-ELISA (cellular ELISA) for the determination of very low levels of IgG on the human erythrocyte membrane has been developed. This CELL-ELISA is based on the use of biotin conjugated to goat anti-human IgG (GaHIgG) and streptavidin conjugated to alkaline phosphatase. The CELL-ELISA can be accurately performed on either fresh or glutaraldehyde fixed erythrocytes. When a population of healthy young adults was studied an average of 126 +/- 14 IgG molecules per erythrocyte were detected.