Flow cytometric determination of FcgammaRIIa (CD32) polymorphism.
A guanine to adenine point mutation results in an arginine (R) to histidine (H) substitution in FcgammaRIIa at residue 131 that strongly impacts receptor function. This FcgammaRIIa 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 FcgammaRII, and one of them, MDE-9, selectively recognized the FcgammaRIIa-H131 allotype. MDE-9 was applicable to detect FcgammaRIIa-H131 in both flow cytometry and immunohistochemistry. MDE-9 was used to develop an FcgammaRIIa allotyping method based on flow cytometry. In a "single-tube assay", FITC-labeled MDE-9 (specific for FcgammaRIIa-H131) and Cy3-labeled mAb 41H16 (specific for FcgammaRIIa-R131) were added to 50 mul samples of whole blood. The results of flow cytometric FcgammaRIIa 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 FcgammaRIIa-H/H131 homozygous individuals to detect FcgammaRIIa and FcgammaRIIb 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 FcgammaRIIb.
van Royen-Kerkhof
Annet
A
Immunotherapy Laboratory, Department of Immunology, KC 02.085.2, University Medical Center Utrecht, Lundlaan 6, 35484 EA, Utrecht, The Netherlands.
Sanders
Elisabeth A M
EA
Wijngaarden
Siska
S
van Roon
Joel A G
JA
Voorhorst-Ogink
Marleen
M
Walraven
Vanessa
V
Gerritsen
Arnout
A
van Dijk
Marc A
MA
Kuis
W
W
Rijkers
Ger T
GT
Keler
Tibor
T
Leusen
Jeanette H W
JH
van de Winkel
Jan G J
JG
D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D005838:Genotype; D006639:Histidine / Q000235:genetics / Q000276:immunology; D006720:Homozygote; D006801:Humans; D007150:Immunohistochemistry; D019169:Jurkat Cells; D051379:Mice; D017354:Point Mutation / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000276:immunology*; D017452:Receptors, IgG / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection
D000906:Antibodies; D015703:Antigens, CD; C080568:Fc gamma receptor IIA; D017452:Receptors, IgG; D006639:Histidine; D001120:Arginine