We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors—providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.
Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 μm fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple FcγRs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of FcγRs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.
The assay described here allows high-throughput analysis of the phagocytic activity of clinical antibody samples by utilizing a monocytic cell line long utilized to study phagocytic processes10 and plate based automated flow cytometry. This assay is advantageous over others in its ability to precisely characterize antigen-specific antibody subsets, allowing not only for study of differences in phagocytosis driven by disease-specific antibodies from different subjects, but also study of multiple antigen specificities within the same subject. Because antibody recruitment of innate effector cells, including monocytes and other antigen presenting cells strongly impacts disease outcome11-13, and is biologically variable depending antibody geometry, IgG subclass, and glycosylation at Asparagine297 of the Fc domain14-16, this method provides for evaluation of a critical antibody mechanism of action. Furthermore, because antibody-mediated phagocytosis is exploited by some pathogens during the course of infection7,17,18, this assay holds promise in evaluation of the process of antibody-dependent enhancement, and highlights the dual nature of phagocytosis as a potentially protective, as well as potentially detrimental antibody activity.
The assay described can be utilized to analyze antibodies from clinical samples of patient populations, but also of vaccinees, and may be adapted to utilize serum rather than purified IgG. Additionally, cytokine secretion in response to phagocytosis can be analysed from the culture supernatant, and the influence of specific FcR can be determined by using FcR-blocking antibodies, allowing not only differences in phagocytic potency to be determined, but downstream signaling events, with insight into mechanism, and may help to resolve and refine understanding of this immune mechanism.
The authors have nothing to disclose.
The authors wish to acknowledge funding from NIH 3R01AI080289-02S1, and a Harvard University Center for AIDS Research Scholar Fellowship under NIH/NIAID 2P30AI060354-07.
Name of the reagent | Company | Catalogue number | Comments |
EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit | Thermo Scientfic | 21935 |
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Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-50 membrane | Millipore | UFC905024 | Filter unit size may vary according to antigen size |
FluoSpheres NeutrAvidin labeled microspheres, 1.0 μm, yellow-green fluorescent (505/515) | Invitrogen | F-8776 | Manufacturers produce this product in various colors |
Melon Gel IgG Purification Kit | Thermo | 45212 | Care should be taken to verify the purity of Ab samples by SDS-page. |