A versatile high-throughput assay to characterize antibody-mediated neutrophil phagocytosis

• Published in: Journal of immunological methods Vol. 471; pp. 46 - 56
• Main Authors: Karsten, Christina B., Mehta, Nickita, Shin, Sally A., Diefenbach, Thomas J., Slein, Matthew D., Karpinski, Wiktor, Irvine, Edward B., Broge, Thomas, Suscovich, Todd J., Alter, Galit
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2019; Elsevier
• Subjects: antibodies; Antibodies - immunology; Antibody-dependent neutrophil phagocytosis; Antigen-Antibody Complex - immunology; antigens; Antigens - immunology; blood serum; enzymes; Extracellular Traps - immunology; flow cytometry; Flow Cytometry - methods; High-throughput assay; High-Throughput Screening Assays - methods; Humans; Immune correlates of protection; immunologic receptors; immunomodulators; neutrophils; Neutrophils - immunology; Non-neutralizing antibody; Phagocytes - immunology; phagocytosis; Phagocytosis - immunology; Reproducibility of Results; secretion; Systems serology; Antibody-dependent neutrophil phagocytosis; Non-neutralizing antibody; Immune correlates of protection; Systems serology; High-throughput assay
• ISSN: 0022-1759; 1872-7905; 1872-7905
• DOI: 10.1016/j.jim.2019.05.006

Neutrophils, the most abundant white blood cell, play a critical role in anti-pathogen immunity via phagocytic clearance, secretion of enzymes and immunomodulators, and the release of extracellular traps. Neutrophils non-specifically sense infection through an array of innate immune receptors and inflammatory sensors, but are also able to respond in a pathogen/antigen-specific manner when leveraged by antibodies via Fc-receptors. Among neutrophil functions, antibody-dependent neutrophil phagocytosis (ADNP) results in antibody-mediated opsonization, enabling neutrophils to sense and respond to infection in a pathogen-appropriate manner. Here, we describe a high-throughput flow cytometric approach to effectively visualize and quantify ADNP and its downstream consequences. The assay is easily adaptable, supporting both the use of purified neutrophils or white blood cells, the use of purified Ig or serum, and the broad utility of any target antigen. Thus, this ADNP assay represents a high-throughput platform for the in-depth characterization of neutrophil function. [Display omitted] •A high-throughput antibody-dependent neutrophil phagocytosis (ADNP) assay was developed.•This flow cytometry assay is flexible and can be easily adapted to any pathogen.•Analysis of sample sets by ADNP assay is fast, robust and cost-effective.•Additional neutrophil functions can be profiled in secondary analyses.