Flow cytometry-based phagocytosis assay for sensitive detection of opsonic activity of pneumococcal capsular polysaccharide antibodies in human sera

The development of efficient vaccines against Streptococcus pneumoniae is of major importance for public health. The efficacy of pneumococcal vaccination and induced protection are thought to be reflected by the opsonic antibody titers in sera from vaccines. We describe a novel two-color flow cytometry technique for quantification of antibody-mediated pneumococcal phagocytosis. Serum-opsonised fluorescein-isothiocyanate (FITC)-labelled S. pneumoniae were allowed to attach to neutrophils, split into two aliquots and further incubated either at 4 degrees C (to avoid phagocytosis) or 37 degrees C (to allow phagocytosis). Cell-surface residual opsonic IgG was detected by phycoerythrin (PE)-conjugated anti-human IgG in both samples. The fraction of FITC-labelled bacteria phagocytosed via antibody (F(i)) could be estimated from FITC and PE labels, and reflected the opsonic activity of sera. The technique displayed high sensitivity for the detection of opsonic antibodies, as shown by experiments using pre- and post-immune sera, which documented significantly increased phagocytosis after vaccination, and the observed increase in phagocytosis rates at higher antibody levels. The intrinsic variation of the assay was low, and could be further reduced by the use of effector cells from donors with similar IgG receptor (FcgammaR) allotypes. The method described in this study should be generally applicable to test vaccine efficacy, to evaluate the interaction of bacteria and phagocytes, and to discriminate between antibody-mediated and antibody-independent interactions between bacteria and phagocytes.