The Use of Allele-Specific Recombinant Fcg Receptor IIIb Antigens for the Detection of Granulocyte Antibodies

The Fcg receptor IIIb (FcgRIIIb) for the Fc domain of IgG is expressed exclusively on neutrophils. The FcgRIIIb bears allotypic polymorphisms referred to as NA1, NA2, and SH, which are known for their frequent involvement in alloimmune and autoimmune neutropenias as well as in transfusion reactions. The bactericidal capacity of isolated neutrophils is easily activatable, and activation results in selfdesintegration, thus preventing storage of neutrophils. As a result, only freshly isolated granulocytes can be used for antibody screening, often making it impossible to use typed panel cells. To provide a readily available source of typed panel cells, we therefore established stable mammalian cells expressing recombinant NA1, NA2, and SH antigens. We isolated mRNA from typed neutrophils and then transcribed it in cDNA. The cDNA that codes for the different forms of the FcgRIIIb was amplified by polymerase chain reaction and was subsequently subcloned into the mammalian expression vector pcDNA3. Chinese hamster ovary (CHO) cells were transfected with allele-specific constructs, and stable cell lines expressing FcgRIIIb were selected by flow cytometry. Because human sera show high background fluorescence with transfectants in flow cytometry, the monoclonal antibody–specific isolation of granulocyte antigens (MAIGA) assay was performed. By MAIGA assay, we tested 14 wellcharacterized human alloantibodies directed against the antigens NA1, NA2, and SH; 5 FcgRIIIb-specific isoantibodies; and 12 FcgRIIIb-reactive autoantibodies. Except one NA1and one SH-specific alloantibody, all other antibodies could be identified by the use of CHO transfectants. In contrast to neutrophils, fixed CHO cells can be stored at 4°C for at least 4 weeks or stored frozen for a longer period. This longer shelf life of the transfected CHO cells compared with isolated neutrophils will simplify the detection of the clinically most important FcgRIIIb-reactive alloantibodies and autoantibodies. r 1999 by The American Society of Hematology.