To investigate the correlation between the data that can be obtained using the classical kOPA test and the newly developed fOPA method, we measured fOPA titers in a panel of sera displaying a wide range

of kOPA titers to GBS Ia. Remarkably, a good correlation (R2 = 0.82, p < 0.05) between fOPA and kOPA read outs was observed (Fig. 8). PR-171 mw A subset of sera was also tested against GBS serotype III using the isolate COH1 and a good correlation between the two methods (R2 = 0.85, p < 0.05) was obtained also in this case (data not shown). The data indicate that the fOPA method can be used to test functional antibodies against different serotypes. We developed an opsonophagocytosis assay for GBS using pHrodo™ labeled bacteria. Our method offers several advantages over both killing-based and other fluorescence-based opsonophagocytic assays. The most commonly-used fluorophores in OPA assays are fluorescein (fluorescein, dicarboxyfluorescein, oregon green, dihydrodichlorofluorescein) or Alexa Fluor derivatives. Flow cytometry based on those fluorophores can detect cell-associated fluorescence but cannot distinguish between internalized and adhering bacteria, necessitating quenching steps with trypan blue or Baf-A1 clinical trial ethidium bromide to clean out the background fluorescence of externally bound bacteria.

The pHrodo™-based assay provides sensitive detection without the need for quenching or washings steps, saving time and eliminating measurement uncertainty. Indeed, pHrodo™ is a pH sensitive fluorophore showing a very low fluorescent signal at the neutral pH of extracellular and cytoplasmic environment and a bright fluorescent signal in acidic compartments, such as phago-lysosomes, deriving from Tau-protein kinase the fusion of phagosome-containing bacteria with lysosomes which occurs immediately after internalization. As shown by confocal microscopy images, GBS bacteria labeled with pHrodo™ exhibit low fluorescence outside the cell, yet emit a bright

red fluorescence after internalization into the acidic environment of the phagocyte. By determining whether phagosome containing bacteria mature to phago-lysosome acidic compartments, the pHrodo™ assay is predictive of phagocytic killing. Several different mechanisms can lead to bacterial survival after phagocytosis, rendering the phagocytosis measurement non strictly indicative of pathogen clearance. For instance, it has been observed that certain mycobacteria (e.g. Mycobacterium avium, Mycobacterium tubercolosis) are not always killed even when enclosed in phagocytic cells, because the phagosome-lysosome fusion is not accompanied by the normal acidification that creates the appropriate conditions for killing ( Hornef et al., 2002, Bellaire et al., 2005 and Huynh and Grinstein, 2007). Further, the phagosome-lysosome fusion may not occur or the phagosome may not close.