Detection of human monocyte-reactive alloantibodies by flow cytometry after selective downmodulation of the Fc receptor I

Abstract

Monocyte-reactive human alloantibodies may be of importance in situations such as transfusion reactions and bone marrow and kidney transplantation. So far, only complement-binding monocyte-reactive antibodies can be detected with a cytotoxicity assay. No antiglobulin assays are yet available that also detect noncomplement-fixing monocyte-reactive antibodies. The binding of monomeric IgG with high affinity to the Fc receptor I (FcRI) on monocytes has severely hampered the development of such an assay until now. We report on the selective removal of the FcRI from monocytes to test human sera in a flow cytofluorometry assay for the presence of monocyte-reactive IgG alloantibodies. Selective downmodulation of FcRI was accomplished by incubating the cells with murine monoclonal antibodies against FcRI followed by a second incubation with goat-antimouse IgG polyclonal antibodies. With such modified cells, human complement-binding and noncomplement-binding IgG and IgM alloantibodies against polymorphic determinants of the HLA class I and II glycoproteins, the human monocyte antigen system and polymorphic antigenic determinants of the LFA complex, can be detected in a sensitive and reproducible manner.