Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 357-362
The Use of Allele-Specific Recombinant Fc
Receptor IIIb
Antigens for the Detection of Granulocyte Antibodies
Juergen Bux,
Karin Kissel,
Christine Hofmann, and
Sentot Santoso
From the Institute for Clinical Immunology and Transfusion Medicine,
Justus Liebig University, Giessen, Germany.
The Fc
receptor IIIb (FcRIIIb) for the Fc domain of IgG is
expressed exclusively on neutrophils. The FcRIIIb 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
self-desintegration, 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 FcRIIIb 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 FcRIIIb 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 well-characterized human alloantibodies directed against the antigens
NA1, NA2, and SH; 5 FcRIIIb-specific isoantibodies; and 12 FcRIIIb-reactive autoantibodies. Except one NA1- and 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 FcRIIIb-reactive alloantibodies and autoantibodies.
