Flow cytometric assay for detennination of FcγRIIIA-158 V/F polymorphism

A single nucleotide polymorphism (SNP) of the FCGR3A gene results in two allotypes of Fc gamma receptor IIIA (Fc gamma RIIIA) with valine (V) or phenylalanine (F) at amino acid 158. Since the Fc gamma RIIIA-158 V/F polymorphism is associated with the efficacy of monoclonal antibody (mAb) treatment and a risk factor for autoimmune disease, widely applicable methods to assess the SNP are needed. We developed a novel flow cytometric test for this polymorphism using a mAb that recognized only the Fc gamma RIIIA-158 V allele (MEM-154) together with a mAb that detected both Fc gamma RIIIA-158 alleles (3G8). The expression of both Fc gamma RIIIA epitopes on natural killer (NK) cells from 37 healthy donors were measured and compared to the FCGR3A genotype determined by a 5' nuclease assay. Fc gamma RIIIA expression levels in individuals with identical FCGR3A genotypes varied considerably, resulting in overlapping immunofluorescences by both 3G8 and MEM-154 between Fc gamma RIIIA-158 V/F allotypes. However, the ratio between fluorescences measured using those mAbs in a single individual predicted the FCGR3A genotype with 100% sensitivity and specificity. The novel flow cytometric assay for the Fc gamma RIIIA-158 V/F polymorphism that is based on the MEM-154/3G8 fluorescence ratio requires commercially available reagents and a three-color flow cytometer only. (c) 2005 Elsevier B.V. All rights reserved.