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

The development of efficient vaccines against Streptococcus pneumoniae is o f major importance for public health.. The efficacy of pneumococcal vaccina tion and induced protection are thought to be reflected by the opsonic anti body titers in sera from vaccines. We describe a novel two-color flow cytom etry; technique for quantification of antibody-mediated pneumococcal phagoc ytosis, Serum-opsonised fluorescein-isothiocyanate (FITC)-labelled S. pneum oniae were allowed to attach to neutrophils. split into two aliquots and fu rther incubated either at 4 degreesC (to avoid phagocytosis) or 37 degreesC (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 estimat ed from FITC and PE labels, and reflected the opsonic activity of sera. The technique displayed high sensitivity for the detection of opsonic antibodi es, as shown by experiments using pre- and post-immune sera, which document ed significantly increased phagocytosis after vaccination, and the observed increase in phagocytosis rates at higher antibody levels. The intrinsic va riation of the assay was low, and could be further reduced by the use of ef fector cells from donors with similar IBG receptor (Fc gammaR) allotypes, T ile method described in this study should be generally applicable to test v accine efficacy, to evaluate the interaction of bacteria and phagocytes, an d to discriminate between antibody-mediated and antibody-independent intera ctions between bacteria and phagocytes. (C) 2001 Elsevier Science B.V. All rights reserved.