A pathogen-free flow cytometry based opsonophagocytosis assay protocol to quantify antibody-mediated phagocytosis

Abstract

An important function of antibodies is their ability to mark cells for phagocytosis and subsequent degradation in a process known as opsonization. When developing new vaccine candidates, it is critical to determine whether the antigen specific antibodies elicited in response to immunization have this function. Due to the high containment working constraints of several important human pathogens, it is not always possible to directly study this property using the pathogen of interest. Therefore, we sought to develop a pathogen-free, flow cytometry based assay to quantify the opsonizing activity of antibodies elicited in response to immunization. We studied this using vaccine candidates against Coxiella burnetii, the causative agent of Q-fever that is a potential bioterrorism agent. In our assay, fluorescent polystyrene beads were conjugated to C. burnetii proteins then incubated with the antibody-containing sera. The antibody-coated beads were incubated with macrophages and phagocytosis of the fluorescent beads was assayed by flow cytometry. This assay allows for rapid quantification of the opsonizing capacity of antigen specific antibodies elicited in response to immunization.