Related Experiment Video
Updated: Apr 23, 2026
Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing
Published on: March 15, 2019
Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques
Ksenia Matlawska-Wasowska1, James M Gale, Christian K Nickl
1Department of Pediatrics, Division of Hematology and Oncology, MSC 10 5590, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA, KMatlawska-Wasowska@salud.unm.edu.
Background:
Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP.
Objective:
The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes.
Methods:
We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches.
Results:
The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes.
Conclusion:
Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.
