Flow cytometric assay for determination of FcgammaRIIIA-158 V/F polymorphism.
A single nucleotide polymorphism (SNP) of the FCGR3A gene results in two allotypes of Fcgamma receptor IIIA (FcgammaRIIIA) with valine (V) or phenylalanine (F) at amino acid 158. Since the FcgammaRIIIA-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 FcgammaRIIIA-158 V allele (MEM-154) together with a mAb that detected both FcgammaRIIIA-158 alleles (3G8). The expression of both FcgammaRIIIA epitopes on natural killer (NK) cells from 37 healthy donors were measured and compared to the FCGR3A genotype determined by a 5' nuclease assay. FcgammaRIIIA expression levels in individuals with identical FCGR3A genotypes varied considerably, resulting in overlapping immunofluorescences by both 3G8 and MEM-154 between FcgammaRIIIA-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 FcgammaRIIIA-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.
Böttcher
Sebastian
S
Second Department of Medicine, University of Schleswig-Holstein, Campus Kiel, Chemnitzstrasse 33, D-24116 Kiel, Germany. s.boettcher@med2.uni-kiel.de
Ritgen
Matthias
M
Brüggemann
Monika
M
Raff
Thorsten
T
Lüschen
Silke
S
Humpe
Andreas
A
Kneba
Michael
M
Pott
Christiane
C
D000483:Alleles; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000939:Epitopes / Q000032:analysis / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005838:Genotype; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D010649:Phenylalanine / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017452:Receptors, IgG / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D014633:Valine / Q000235:genetics
D000911:Antibodies, Monoclonal; D000939:Epitopes; C496124:FCGR3A protein, human; D017452:Receptors, IgG; D010649:Phenylalanine; D014633:Valine