Cell Based Assays
CD16 (FcγRIII) Phenotyping Services to Determine V/F Polymorphism
In ADCC, natural killer (NK) cells, neutrophils and macrophages bind antibody-coated cells by receptors that recognize the constant region of immunoglobulin (Fcγ receptors, FcγRs). CD16 (FcγRIII) is a low affinity Fc receptor expressed on the surface of NK cells. Binding mediated by the FcγRIIIA receptor(CD16A, encoded by FCGR3A) plays an important role in NK cell activation. Cell Technology Inc. offers CD16 phenotyping services based on allelic polymorphism in NK cell FcγRIIIA at Phe158/Val158 which may influence affinity of Mab Fc-binding and antibody therapeutic efficacy.
We developed a flow cytometry-based test for this polymorphism using a mAb that recognized only the FcγRIIIA-158 V allele (antibody 1) in combination with a mAb that detected both FcγRIIIA-158 alleles (antibody 2). The ratio between the mean fluorescence intensity (MFI) measured with those mAbs (Table 1) in a single individual predicted the FCGR3A genotype with 100% sensitivity and specificity.
The service provides highly customized solutions designed to meet your specific research needs. It is a considerably faster, efficient and cost-effective phenotyping program which enables broad spectrum analysis of the specific ADCC responses in patients and the therapeutic efficacy of antibody drugs.
Figure 1. Flow cytometric gating strategy for determination of CD 16 (FcγRIIIA-158) V/F polymorphism. (a, b): NK cells are gated from PBMC and CD 56+ CD3-region. (c – h): CD 16 fluorescence of NK cells from three individuals genotyped as FcγRIIIA-158 F /F (c, f), V/F (d, g), and V/V (e, h). NK cells were labeled with CD 16 antibody 1 or antibody 2 and counter stained with FITC conjugated Goat anti Mouse antibodies. The MFI ratios of antibody 1/2 were F/F: c/f < 0.04, V/F: d/g = 0.42, and V/V: e/h = 0.83, respectively.
Table 1. Standard used to determine CD16 Phenotypes:
Steps Involved for implementation
Obtaining fresh blood samples from random donors.
Isolate PBMC’s from blood samples.
Stain cells with antibodies for V and V/F allele visualization.
Determine immunofluorescence ratio for antibody 1 and antibody 2 to determine V/V, V/F or F/F phenotype by Flow cytometer.
Supplies and information to be provided by the client
Numbers of donors to be tested.
10 ml of Blood samples or 1ml of PBMC cells for each donor ( 1x 106 cells/ml).
Supplies to be provided by Cell Technology
All additional supplies would be procured by Cell Technology, and billed to the client at cost.
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