Flow cytometric determination of FcγRIIa (CD32) polymorphism

Abstract

A guanine to adenine point mutation results in an arginine (R) to histidine (H) substitution in FcγRIIa at residue 131 that strongly impacts receptor function. This FcγRIIa polymorphism is mostly typed by allele-specific polymerase chain reactions (PCR) or in functional assays, dependent on ligand binding. Both types of methods are laborious, time consuming, and not readily available in routine laboratories. We generated a panel of human antibodies against FcγRII, and one of them, MDE-9, selectively recognized the FcγRIIa-H131 allotype. MDE-9 was applicable to detect FcγRIIa-H131 in both flow cytometry and immunohistochemistry. MDE-9 was used to develop an FcγRIIa allotyping method based on flow cytometry. In a "single-tube assay", FITC-labeled MDE-9 (specific for FcγRIIa-H131) and Cy3-labeled mAb 41H16 (specific for FcγRIIa-R131) were added to 50 μl samples of whole blood. The results of flow cytometric FcγRIIa allotyping correlated completely with PCR genotyping. This novel allotyping assay should facilitate the screening of patients in a routine diagnostic setting. In addition, a combination of MDE-9 and 41H16 can be used in FcγRIIa-H/H131 homozygous individuals to detect FcγRIIa and FcγRIIb surface expression on monocytes. This is an important application of these antibodies because, to this day, no antibodies were available to specifically study the surface expression of FcγRIIb.